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Time Course and Site(s) of Cytochrome c Tyrosine Nitration by Peroxynitrite †
Abstract
Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.
... In the native configuration, the heme possesses an absorption band centered at 695 nm (16). Under relevant cellular conditions, such as interactions with different phospholipids (cardiolipin) (17)(18)(19)(20)(21)(22)(23), electric fields (24,25), or amino acid oxidative PTMs (tyrosine nitration and methionine oxidation) (26)(27)(28)(29), cyt c can give rise to nonnative, alternative protein conformations (24,25). All these alternative conformations share a gain-of-peroxidase activity and the disruption of the heme ligand at the sixth axial M80-Fe position, causing a loss of the absorption at 695 nm (17-19, 23, 27, 30, 31). ...
... Tyr74 (Y74) revealed that nitration causes a conformational transition at physiological pH (26,28,30,32). Indeed, in NO 2 -Tyr74 cyt c, the conformational change known as "alkaline transition" (33)(34)(35) shifts its pKa from 9.2 to 7.2 (26). ...
... Since the disruption of the M80-Fe bond is observed during the alkaline transition of cyt c, when bound to phospholipids (20)(21)(22)(23), and also when cyt c is treated with different oxidants (peroxynitrite, HOCl, and H 2 O 2 ) (26,27), we aimed to measure the ability of mAb 1D3 and R1D3 to recognize site-specific mononitrated (NO 2 Y74 and NO 2 Y97) and methionine-sulfoxidized (SO-M80) cyt c proteoforms that also lack the Fe-M80 ligation. The gain-of-peroxidase activity was also checked for these conformations, and all of them were shown to be increased (SI Appendix, Fig. S4), as previously reported (28,37). Purified NO 2 Y74 and NO 2 Y97 as well as SO-M80 cyt c species were used in competitive ELISA (Fig. 3). ...
Cytochrome c (cyt c ) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping–mass spectrometry–bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40–57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.
... In particular, the incorporation of a nitro-group in the side chain results in a drop of the pKa of the phenolic hydroxyl group from about 10 to 6.8-7.2, leading to its ionization and therefore an additional negative charge at physiologically-relevant pH [28][29][30]. The influence of these physico-chemical changes in tyrosine on the capability of the proteasome to handle tyrosine-containing peptides is far from obvious. ...
... Since horse Cyt c is a small protein, containing only four tyrosine (Y) residues, modification by tyrosine nitration and peptide sequence analyses after proteasome digestion is much easier to perform. In this sense, we have previously characterized the peroxynitrite-dependent formation of tyrosine nitrated Cyt c species (NO 2 Y-Cyt c) and developed protocols for the separation and purification of site-specific tyrosine nitrated Cyt c proteoforms [30,32]. Interestingly, we and others have shown that nitro-oxidative stress to cells leads NO 2 Y-Cyt c formation and translocation from the mitochondria into the cytosol and nucleus [33][34][35]. ...
... In order to perform initial degradation studies with the isolated proteasome, native, H 2 O 2 -oxidized and two main (mono)-nitrated Cyt c proteoforms were used. These two nitrated Cyt c (NO 2 -Cyt c) species were prepared, isolated and purified according to previous protocols of our group [30] (SI Appendix, Fig. 1A and B) and consist in a fraction composed mainly by Cyt c in which Y74 is nitrated (NO 2 Y74-Cyt c) and a second fraction composed by a Cyt c species in which Y97 is nitrated (NO 2 Y97-Cyt c). Immunochemical characterization of the two purified fractions revealed the presence of NO 2 Y (SI Appendix, Fig. 1C) in the two Cyt c proteoforms. ...
Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicates that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded and facilitating, the accumulation of nitrated proteins.
... In particular, peroxynitrite anion is one of the most relevant agents causing tyrosine nitration in vivo [39][40][41], which occurs mainly by two mechanisms, direct reaction of the oxidant with a target or via the product of it homolysis, nitrogen dioxide and hydroxyl radical [42]. Peroxynitrite-mediated tyrosine nitration 3 of cytochrome c has been reported and may lead to conformational changes and enhanced peroxidatic activity as well [4,26,[43][44][45][46][47]. Cytochrome c possesses four highly conserved tyrosines at positions 48, 67, 74 and 97 and, in particular, the gain-of-peroxidatic activity is associated with Tyr74 nitration that triggers an early "alkaline transition" [38]. ...
... Cytochrome c possesses four highly conserved tyrosines at positions 48, 67, 74 and 97 and, in particular, the gain-of-peroxidatic activity is associated with Tyr74 nitration that triggers an early "alkaline transition" [38]. Previous studies did not find a direct reaction of the cytochrome c with peroxynitrite but rather tyrosine nitration is mediated by peroxynitrite-derived radicals [47][48][49]. ...
... Even though the reaction of peroxynitrite with cytochrome c generates a collection of tyrosine nitrated cyt c proteoforms [36,50], in solution the changes occur preferentially at Tyr74 and 97 [47]. Kapralov et al. [51] showed that the heme adjacent Tyr67 (Table 2) could have an important influence in the peroxidatic activity of cytochrome c upon interaction with cardiolipin. ...
The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis.
... Among the better studied posttranslational modifications (Table 1), the nitration of cyt c by peroxynitrite and other nitric oxide-derived oxidants has received an increasing amount of attention, largely because of its potential role in human diseases accompanied by oxidative stress. 29,123 Of the four conserved Tyr residues of human cyt c, Tyr74 and Tyr97 are exposed to the solvent whereas Tyr67 and Tyr48 are buried within the protein matrix ( Figure 9). Monoand dinitrated variants of cyt c can be prepared in vitro using slow fluxes of peroxynitrite near physiological conditions and purified to homogeneity ( Figure 10). ...
... Monoand dinitrated variants of cyt c can be prepared in vitro using slow fluxes of peroxynitrite near physiological conditions and purified to homogeneity ( Figure 10). 123,124 Nitration of Tyr residues occurs in vivo and alters the functions of proteins. 125−127 Endogenously formed nitrated cyt c has been detected in cultured cells and in disease models characterized by nitroxidative stress. ...
... 59 While the dynamics of H-bond formation via residue Tyr67 may be promiscuous in nature, consensus about the importance of far-reaching effects in controlling heme electronics and, therefore, the peroxidase activity of cyt c exists. Individual, successive replacement of Tyr with Phe leads to additive effects in reducing the midpoint potential of cyt c, even though no correlation could be established between the previously reported values of peroxidase activity 123,138 and the relative contribution of each non-native coordination state. 135 This lack of correlation is not surprising per se, as RR measurements do not report on the conformational changes that accompany the different coordination states within the alkaline ensemble. ...
Cytochrome c (cyt c) is a cationic hemoprotein of ca. 100 amino acid residues that exhibits exceptional functional versatility. While its primary function is electron transfer in the respiratory chain, cyt c is also recognized as a key component of the intrinsic apoptotic pathway, the mitochondrial oxidative protein folding machinery and presumably as a redox sensor in the cytosol, along with other reported functions. Transition to alternative conformations and gain-of-peroxidase activity are thought to further enable the multiple functions of cyt c and its translocation across cellular compartments. In vitro, direct interactions of cyt c with cardiolipin, post-translational modifications such as tyrosine nitration, phosphorylation, methionine sulfoxidation, mutations and even fine changes in electrical fields lead to a variety of conformational states that may be of biological relevance. The identification of these alternative conformations and the elucidation of their function in vivo continue to be a major challenge. Here, we unify knowledge on the structural flexibility of cyt c that supports functional moonlighting and review biochemical and immunochemical evidence confirming that cyt c undergoes conformational changes during normal and altered cellular homeostasis.
... Nitration affects the redox potential of Cc as well as its electron-exchange kinetics, depending on the residue involved [203]. The nitration of Y46, Y48, Y74, and Y97 residues also increases the peroxidase activity of Cc and lowers the pK A value of the alkaline transition besides other functional properties [179,180,183,185,197,204,205] (Table 1). Nitration of Cc has been associated with several diseases, including chronic nephropathy [206]. ...
... All modifications/mutations of S47 and Y67 alter the peroxidase activity of Cc [79,179,185,192,195,197]. Y67 is located close to Met80 and is part of the hydrophobic pocket which houses the acyl chains of CL. ...
Cardiolipin oxidation and degradation by different factors under severe cell stress serve as a trigger for genetically encoded cell death programs. In this context, the interplay between cardiolipin and another mitochondrial factor—cytochrome c—is a key process in the early stages of apoptosis, and it is a matter of intense research. Cytochrome c interacts with lipid membranes by electrostatic interactions, hydrogen bonds, and hydrophobic effects. Experimental conditions (including pH, lipid composition, and post-translational modifications) determine which specific amino acid residues are involved in the interaction and influence the heme iron coordination state. In fact, up to four binding sites (A, C, N, and L), driven by different interactions, have been reported. Nevertheless, key aspects of the mechanism for cardiolipin oxidation by the hemeprotein are well established. First, cytochrome c acts as a pseudoperoxidase, a process orchestrated by tyrosine residues which are crucial for peroxygenase activity and sensitivity towards oxidation caused by protein self-degradation. Second, flexibility of two weakest folding units of the hemeprotein correlates with its peroxidase activity and the stability of the iron coordination sphere. Third, the diversity of the mode of interaction parallels a broad diversity in the specific reaction pathway. Thus, current knowledge has already enabled the design of novel drugs designed to successfully inhibit cardiolipin oxidation.
... Human Cytc has 5 tyrosine residues at sites 46,48,67,74, and 97, of which 4 are evolutionarily conserved in mammals (61,124). Of the 5 tyrosine residues, only 3 (Tyr67, Tyr74, and Tyr97) are nitrated in vitro in the presence of peroxynitrite (125). Tyr67-and Tyr74nitrated Cytc are less efficient in restoring respiratory capacity in Cytc-deficient mitochondria compared to Tyr97 (125,126). ...
... Of the 5 tyrosine residues, only 3 (Tyr67, Tyr74, and Tyr97) are nitrated in vitro in the presence of peroxynitrite (125). Tyr67-and Tyr74nitrated Cytc are less efficient in restoring respiratory capacity in Cytc-deficient mitochondria compared to Tyr97 (125,126). Tyr48, which is highly conserved, and Tyr46, which is present only in humans and plants, were the least commonly nitrated residues (127). ...
Cytochrome c (Cytc) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cytc has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cytc is tightly regulated by allosteric mechanisms, tissue‐specific isoforms, and post‐translational modifications (PTMs). Distinct residues of Cytc are modified by PTMs, primarily phosphorylations, in a highly tissue‐specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨm), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia–reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨm hyperpolarization, excessive ROS generation, and the release of Cytc. It is also the dephosphorylated form of the protein that leads to maximum caspase activation. We discuss the complex regulation of Cytc and propose that it is a central regulatory step of the mammalian ETC that can be rate limiting in normal conditions. This regulation is important because it maintains optimal intermediate ΔΨm, limiting ROS generation. We examine the role of Cytc PTMs, including phosphorylation, acetylation, methylation, nitration, nitrosylation, and sulfoxidation and consider their potential biological significance by evaluating their stoichiometry.—Kalpage, H. A., Bazylianska, V., Recanati, M. A., Fite, A., Liu, J., Wan, J., Mantena, N., Malek, M. H., Podgorski, I., Heath, E. I., Vaishnav, A., Edwards, B. F., Grossman, L. I., Sanderson, T. H., Lee, I., Hüttemann, M. Tissue‐specific regulation of cytochrome c by post‐translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis. FASEB J. 33, 1540–1553 (2019). www.fasebj.org
... Among these, the most deeply studied is the nitration of the various Tyr residues present in cyt c, under conditions relevant to pathologies associated with oxidative stress. 715,716 hh-Cyt c possesses four Tyr residues of which Y74 and Y97 are exposed to the solvent, Y67 is buried within the protein skeleton, and Y48 exhibits enough solvent accessibility to undergo phosphorylation 33 (Figure 31). Methods for the production and isolation of mono and dinitrated variants of cyt c have been developed using low fluxes of peroxynitrite and tetranitromethane. ...
... 193 While formation and rupture of H-bonding interactions via residue Y67 may be a highly dynamic event, it is broadly accepted that far-reaching effects control heme electronics and, therefore, the peroxidase activity of cyt c. Resonance Raman studies investigating individual and successive substitutions of Tyr by Phe residues demonstrated additive effects in redox potential, but no direct correlation with previously reported values of peroxidase activity 715,728 or with the relative contribution of each non-native conformer in these mutagenic variants. 727 It is important to know that this apparent lack of correlation may not accurately reflect the authentic behavior of each variant, as the technique itself does not measure the conformational changes that accompany each coordination state within the alkaline ensemble. ...
Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein.
... Interaction of cyt c with anionic phospholipids and/or reactions with oxidants, such as peroxynitrite, increases the peroxidase activity of cyt c [219,220,224]. In fact, biologically relevant concentrations of peroxynitrite have been shown to promote cyt c release from intact mitochondria [219,[224][225][226]. It has been proposed that • NO may regulate the peroxidase activity of cyt c through reduction of highly oxidized states of the heme [224,227]. ...
... This posttranslational modification affects cyt c redox properties impairing its electron transport ability in the mitochondrial respiratory chain. Indeed, basal respiration cannot be recovered when cyt c-depleted mitochondria are reconstituted with peroxynitrite-treated cyt c [224,226]. ...
It has long been recognized that energy metabolism is linked to the production of reactive oxygen species (ROS) and critical enzymes allied to metabolic pathways can be affected by redox reactions. This interplay between energy metabolism and ROS becomes most apparent during the aging process and in the onset and progression of many age-related diseases (i.e. diabetes, metabolic syndrome, atherosclerosis, neurodegenerative diseases). As such, the capacity to identify metabolic pathways involved in ROS formation, as well as specific targets and oxidative modifications is crucial to our understanding of the molecular basis of age-related diseases and for the design of novel therapeutic strategies.
Herein we review oxidant formation associated with the cell's energetic metabolism, key antioxidants involved in ROS detoxification, and the principal targets of oxidant species in metabolic routes and discuss their relevance in cell signaling and age-related diseases.
... While NK1 contains six tyrosine residues (Fig. S1 panel B), not all tyrosine residues within a protein are nitrated [2,45,60]. Several factors may be involved in tyrosine nitration selectivity: exposure of the tyrosine aromatic-ring to the protein surface may facilitate the initial attack by peroxynitrite while the probability for nitration of buried tyrosine residues is low [61]. The residues in the vicinity of a tyrosine residue are also critical for its possible nitration: a tyrosine close to a glutamate has a high tendency for nitration [62]. ...
Protein tyrosine residue (Y) nitration, a post-translational chemical-modification mode, has been associated with changes in protein activity and function; hence the accumulation of specific nitrated proteins in tissues may be used to monitor the onset and progression of pathological disorders. To verify the possible impact of nitration on postnatal muscle growth and regeneration, a pilot study was designed to examine the nitration/dysfunction of hepatocyte growth factor (HGF), a key ligand that is released from the extracellular tethering and activates myogenic stem satellite cells to enter the cell cycle upon muscle stretch and injury. Exposure of recombinant HGF (a hetero-dimer of α- and β-chains) to peroxynitrite induces Y nitration in HGF α-chain under physiological conditions. Physiological significance of this finding was emphasized by Western blotting that showed the NK1 segment of HGF (including a K1 domain critical for signaling-receptor c-met binding) undergoes nitration with a primary target of Y198. Peroxynitrite treatment abolished HGF-agonistic activity of the NK1 segment, as revealed by in vitro c-met binding and bromodeoxyuridine-incorporation assays. Importantly, direct-immunofluorescence microscopy of rat lower hind-limb muscles from two aged-groups (2-month-old "young" and 12-month-old "retired/adult") provided in vivo evidence for age-related nitration of extracellular HGF (Y198). Overall, findings provide the insight that HGF/NK1 nitration/dysfunction perturbs myogenic stem cell dynamics and homeostasis; hence NK1 nitration may stimulate progression of muscular disorders and diseases including sarcopenia.
... While NK1 contains six tyrosine residues (Fig. S1 panel B), not all tyrosine residues within a protein are nitrated [2,45,60]. Several factors may be involved in tyrosine nitration selectivity: exposure of the tyrosine aromatic-ring to the protein surface may facilitate the initial attack by peroxynitrite while the probability for nitration of buried tyrosine residues is low [61]. The residues in the vicinity of a tyrosine residue are also critical for its possible nitration: a tyrosine close to a glutamate has a high tendency for nitration [62]. ...
... low % modification present. To address this limitation, other authors employed a capture step with antibodies against 3-nitrotyrosine [22,50,51], or cation-exchange HPLC [52] for the enrichment of nitrated proteins or nitrated peptides before LC-MS/MS analysis but, as mentioned above, this can introduce bias in the analysis. In contrast, we used a targeted MS/MS analysis to improve sensitivity, including transitions to detect modifications observed in the lowest peroxynitrite treatment (Y62 from the α chain, Y71, Y408 and Y475 from β chain, and Y389 from γ chain) as well as several others there were consistently nitrated (277 and Y589 from α chain, Y452 from β chain and Y380 from γ chain). ...
Ischemic stroke is one of the leading causes of death and disability worldwide. This acute vascular event interferes with blood supply to the brain and induces a burst of free radicals such as nitric oxide and superoxide, producing peroxynitrite, a precursor of strong nitrating agents. Fibrinogen is one of the most abundant plasma proteins; it plays a role in the hemostatic system, mediating clot formation, which can be affected by nitrotyrosine formation. We hypothesized that nitration of fibrinogen by ONOOH and ONOOCO2- radical products could be one of the early events of the ischemic stroke, and protein-bound 3-nitrotyrosine could be a potential biomarker for diagnosis and/or prognosis of this condition. A targeted mass spectrometry approach was developed to analyze the nitration of fibrinogen and its association with ischemic stroke. First, a comprehensive mapping of 3-nitrotyrosine locations and their relative quantification was performed by LC-MS/MS, using in vitro nitrated fibrinogen samples. Twenty different 3-nitrotyrosine residues were identified on fibrinogen nitrated in vitro, varying with the peroxynitrite: fibrinogen molar ratio used. Nine tyrosine residues that were consistently modified at different treatment ratios were chosen to perform a targeted LC-MS/MS analysis in clinical samples. Enriched fibrinogen fractions from clinical samples from 24 ischemic stroke and 12 patients with non-inflammatory conditions were analyzed with this method. Three of the nine tyrosine residues analyzed (βY452, βY475 and γY380) showed a significant difference between the ischemic stroke and non-inflammatory disease groups. ROC curve analysis suggested an association of these residues either individually or in combination with ischemic stroke. Different tyrosine nitration patterns were also observed in fibrinogen modified in vitro and in vivo, suggesting differences in the nitration process in these situations. This is the first study showing a putative association between the nitration profile of specific tyrosine residues in human fibrinogen and ischemic stroke.
... The reported cases of proteins which experience a gain of function due to tyrosine nitration are, by far, much less. A well-described example is the case of cytochrome c, which undergoes a conformational change after nitration of Tyr74, adopting an alternative conformation that has an increased peroxidatic activity [166][167][168]. As for MnSOD, this effect is a direct consequence of the properties of NO 2 Tyr: nitration promotes the ionization of Tyr74 at physiological pH, which in turn triggers the conformational change [169]. ...
Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical–radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO−), the product of the reaction of superoxide radical (O2•−) with •NO, but ONOO−-independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.
... The mutants Y67F and E66Q, as well as the variants WT Cyt c and Y67F nitrated at Tyr74 (NO 2 -Cyt c and NO 2 -Y67F, respectively) were produced and purified following published procedures [11,39]. ...
Cytochrome c is a prototypical multifunctional protein that is implicated in a variety of processes that are essential both for sustaining and for terminating cellular life. Typically, alternative functions other than canonical electron transport in the respiratory chain are associated to alternative conformations. In this work we apply a combined experimental and computational study of Cyt c variants to assess whether the parameters that regulate the canonical electron transport function of Cyt c are correlated with those that determine the transition to alternative conformations, using the alkaline transition as a model conformational change. The results show that pKa values of the alkaline transition correlate with the activation energies of the frictionally-controlled electron transfer reaction, and that both parameters are mainly modulated by the flexibility of the Ω-loop 70-85. Reduction potentials and non-adiabatic ET reorganization energies, on the other hand, are both modulated by the flexibilities of the Ω-loops 40-57 and 70-85. Finally, all the measured thermodynamic and kinetic parameters that characterize both types of processes exhibit systematic variations with the dynamics of the hydrogen bond between the axial ligand Met80 and the second sphere ligand Tyr67, thus highlighting the critical role of Tyr67 in controlling canonical and alternative functions of Cyt c.
... [143,144] Brain energy levels can be affected in people with schizophrenia by NO's effect on mitochondrial activity, ATP production, [145] and the formation of peroxynitrite, which causes oxidative damage to mitochondrial structural proteins and enzymes as well as damage to membrane lipids. [14,146,147] Most pharmacological treatments for schizophrenia treatment suppress dopamine, but medications are not effective for all patients and are associated with side effects, which may be severe. [148] The majority of patients with schizophrenia are obese, [149] although the possible association of schizophrenia and obesity remains to be elucidated. ...
... In MALDI-based procedures for peptide and protein analysis, 3-nitrotyrosine residues absorb the light of typical lasers (337 nm) and produce a characteristic pattern of ions in which the m/z is decreased by 16 and 30. The unique triplet signal allows to further identify the nitrated peptides (243,244). Nevertheless, methods based on electrospray ionization present the advantages that photochemical decomposition is avoided and that the typical ion with m/z 181 can be detected and serve as footprint for 3-nitrotyrosine (221). ...
The free radical nitric oxide (NO•) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO•-derived reactive species are typically short-lived and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this review, we describe the biologically relevant characteristics of the reactive species formed downstream from NO•, and we discuss the approaches currently available for the analysis of NO•, nitrogen dioxide (NO2•), dinitrogen trioxide (N2O3), nitroxyl (HNO) and peroxynitrite (ONOO-/ONOOH), as well as peroxynitrite-derived hydroxyl (HO•) and carbonate anion (CO3•-) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2-), nitrate (NO3-), nitrosyl-metal complexes, S-nitrosothiols and 3-nitrotyrosine. Moreover, we highlight state-of-the art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO•-derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involved.
... Disulfide-oxidized Prx2 (130 μM) was treated with a five-fold excess peroxynitrite in a flux-like addition, as described in Ref. [19]. This protocol had to be followed rigorously in order to obtain reproducible results after nitration, considering that nitration yields depend on the rate of radicals production from peroxynitrite [21,40]. As a control, peroxynitrite was previously decomposed in the assay buffer and then added to the protein (reverse-order addition, ROA). ...
Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.
... Nitric oxide can also serve as a source of reactive nitrogen species (RNS). Overaccumulation of RNS under abiotic stress can cause tyrosine nitration and inactivate proteins like CAT, manganese-dependent (Mn-)SOD, and GR as well as the peroxidative activity of cytochrome c [37,38]. Our results showed that NO 3 − -supplied plants had more severe oxidative damage and accumulated extremely high NO levels after 24 h of water stress (Fig. 3). ...
Background
Nutrition with ammonium (NH4⁺) can enhance the drought tolerance of rice seedlings in comparison to nutrition with nitrate (NO3⁻). However, there are still no detailed studies investigating the response of nitric oxide (NO) to the different nitrogen nutrition and water regimes. To study the intrinsic mechanism underpinning this relationship, the time-dependent production of NO and its protective role in the antioxidant defense system of NH4⁺- or NO3⁻-supplied rice seedlings were studied under water stress.
Results
An early NO burst was induced by 3 h of water stress in the roots of seedlings subjected to NH4⁺ treatment, but this phenomenon was not observed under NO3⁻ treatment. Root oxidative damage induced by water stress was significantly higher for treatment with NO3⁻ than with NH4⁺ due to reactive oxygen species (ROS) accumulation in the former. Inducing NO production by applying the NO donor 3 h after NO3⁻ treatment alleviated the oxidative damage, while inhibiting the early NO burst by applying the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) increased root oxidative damage in NH4⁺ treatment. Application of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester(L-NAME) completely suppressed NO synthesis in roots 3 h after NH4⁺ treatment and aggravated water stress-induced oxidative damage. Therefore, the aggravation of oxidative damage by L-NAME might have resulted from changes in the NOS-mediated early NO burst. Water stress also increased the activity of root antioxidant enzymes (catalase, superoxide dismutase, and ascorbate peroxidase). These were further induced by the NO donor but repressed by the NO scavenger and NOS inhibitor in NH4⁺-treated roots.
Conclusion
These findings demonstrate that the NOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by water stress by enhancing the antioxidant defenses in roots supplemented with NH4⁺.
... presence of CO 2 as observed for Y48, Y74 and Y97 of cytochrome c [88]. The redox environment of the particular tyrosine residue will determine as well the possibilities of nitration. ...
In this review we provide an analysis of the biochemistry of peroxynitrite and tyrosine nitration. Peroxynitrite is the product of the diffusion-controlled reaction between superoxide (O2(•)(-)) and nitric oxide ((•)NO). This process is in competition with the enzymatic dismutation of O2(•-) and the diffusion of (•)NO across cells and tissues and its reaction with molecular targets (e.g. guanylate cyclase). Understanding the kinetics and compartmentalization of the O2(•-) / (•)NO interplay is critical to rationalize the shift of (•)NO from a physiological mediator to a cytotoxic intermediate. Once formed, peroxynitrite (ONOO(-) and ONOOH; pKa = 6,8) behaves as a strong one and two-electron oxidant towards a series of biomolecules including transition metal centers and thiols. In addition, peroxynitrite anion can secondarily evolve to secondary radicals either via its fast reaction with CO2 or through proton-catalyzed homolysis. Thus, peroxynitrite can participate in direct (bimolecular) and indirect (through secondary radical intermediates) oxidation reactions; through these processes peroxynitrite can participate as cytotoxic effector molecule against invading pathogens and/or as an endogenous pathogenic mediator. Peroxynitrite can cause protein tyrosine nitration in vitro and in vivo. Indeed, tyrosine nitration is a hallmark of the reactions of (•)NO-derived oxidants in cells and tissues and serves as a biomarker of oxidative damage. Protein tyrosine nitration can mediate changes in protein structure and function that affect cell homeostasis. Tyrosine nitration in biological systems is a free radical process that can be promoted either by peroxynitrite-derived radicals or by other related (•)NO-dependent oxidative processes. Recently, mechanisms responsible of tyrosine nitration in hydrophobic biostructures such as membranes and lipoproteins have been assessed and involve the parallel occurrence and connection with lipid peroxidation. Experimental strategies to reveal the proximal oxidizing mechanism during tyrosine nitration in given pathophysiologically-relevant conditions include mapping and identification of the tyrosine nitration sites in specific proteins.
... In particular, nitration of α-synuclein was linked to neurodegeneration in Parkinson's disease and dementia with Lewy bodies, two conditions in which cell death with characteristics of apoptosis were reported (Duda et al., 2000;Giasson et al., 2000;Majd et al., 2015). Cytochrome c is susceptible to nitration by peroxynitrite and other oxidants and nitrated cytochrome c is found in inflammatory conditions (Batthyany et al., 2005;Castro et al., 2004). Nitration of cytochrome c also activates a peroxidase activity that was proposed to play a role in apoptosis by stimulating release of cytochrome c from the mitochondria and the externalization of phosphatidylserine (Kagan et al., 2004). ...
Peroxynitrite (ONOO⁻) is a strong biological oxidant formed by the diffusion-limited reaction of nitric oxide (NO⁻) and superoxide anion (O2⁻). It has long been theorized that peroxynitrite generation could be the cause in a number of pathological conditions ranging from atherosclerosis to inflammatory, autoimmune, heart and neurodegenerative diseases. Its relatively long biological half-life and high reactivity allows peroxynitrite to oxidize a number of different targets in the cell. In physiologically relevant conditions peroxynitrite can directly react with thiols, or the radical products of peroxynitrite decomposition may indirectly oxidize other cellular components such as lipids, proteins and DNA. Downstream, oxidative modifications caused by peroxynitrite trigger cell death by a variety of mechanisms depending on the concentration of the oxidant. Peroxynitrite stimulates necrosis, apoptosis, autophagy, parthanatos and necroptosis. Here we review the mechanisms activated by peroxynitrite to cause neuronal death.
... The succinate-ubiquinone oxidoreductase complex II is relatively resistant to NO or RNS inhibition, but the enzyme iron-sulfur centers are susceptible to irrevocable damage in an environment of chronic O&NS [228,229]. The cytochrome bc 1 complex can be inhibited by excessive levels of NO, but it is unclear whether S-nitrosylation underpins this effect [230,231]. ...
Nitric oxide plays an indispensable role in modulating cellular signaling and redox pathways. This role is mainly effected by the readily reversible nitrosylation of selective protein cysteine thiols. The reversibility and sophistication of this signaling system is enabled and regulated by a number of enzymes which form part of the thioredoxin, glutathione, and pyridoxine antioxidant systems. Increases in nitric oxide levels initially lead to a defensive increase in the number of nitrosylated proteins in an effort to preserve their function. However, in an environment of chronic oxidative and nitrosative stress (O&NS), nitrosylation of crucial cysteine groups within key enzymes of the thioredoxin, glutathione, and pyridoxine systems leads to their inactivation thereby disabling denitrosylation and transnitrosylation and subsequently a state described as "hypernitrosylation." This state leads to the development of pathology in multiple domains such as the inhibition of enzymes of the electron transport chain, decreased mitochondrial function, and altered conformation of proteins and amino acids leading to loss of immune tolerance and development of autoimmunity. Hypernitrosylation also leads to altered function or inactivation of proteins involved in the regulation of apoptosis, autophagy, proteomic degradation, transcription factor activity, immune-inflammatory pathways, energy production, and neural function and survival. Hypernitrosylation, as a consequence of chronically elevated O&NS and activated immune-inflammatory pathways, can explain many characteristic abnormalities observed in neuroprogressive disease including major depression and chronic fatigue syndrome/myalgic encephalomyelitis. In those disorders, increased bacterial translocation may drive hypernitrosylation and autoimmune responses against nitrosylated proteins.
... Peroxynitrite has been shown to react with Prx2 C P , overoxidizing it to its sulfinic and even sulfonic form [9]. To prevent these modifications of C P , treatment with peroxynitrite was performed on the disulfide-oxidized enzyme. The corresponding molar excess of peroxynitrite was added as a flux-like addition that simulate generation of peroxynitrite in vivo (it is worth to note that nitration yields depend on rate of radicals production from peroxynitrite [33,46]). As a control, peroxynitrite was previously decomposed in the assay buffer and then added to the protein (reverse-order addition, ROA). ...
... Thiol oxidation and nitration of tyrosine residues are the major mechanism by which peroxynitrite induces conformational change in proteins [433,434]. Peroxynitrite also causes oxidative damage to mitochondrial structural proteins and enzymes and peroxidative damage to lipids within membranes leading to profound changes in function and membrane integrity [1,435,436]. Peroxynitrite inhibits mitochondrial respiration by inactivation of ETC I and III [437,438]. ...
Mitochondrial dysfunction and defects in oxidative metabolism are a characteristic feature of many chronic illnesses not currently classified as mitochondrial diseases. Examples of such illnesses include bipolar disorder, multiple sclerosis, Parkinson’s disease, schizophrenia, depression, autism, and chronic fatigue syndrome.
While the majority of patients with multiple sclerosis appear to have widespread mitochondrial dysfunction and impaired ATP production, the findings in patients diagnosed with Parkinson’s disease, autism, depression, bipolar disorder schizophrenia and chronic fatigue syndrome are less consistent, likely reflecting the fact that these diagnoses do not represent a disease with a unitary pathogenesis and pathophysiology. However, investigations have revealed the presence of chronic oxidative stress to be an almost invariant finding in study cohorts of patients afforded each diagnosis. This state is characterized by elevated reactive oxygen and nitrogen species and/or reduced levels of glutathione, and goes hand in hand with chronic systemic inflammation with elevated levels of pro-inflammatory cytokines.
This paper details mechanisms by which elevated levels of reactive oxygen and nitrogen species together with elevated pro-inflammatory cytokines could conspire to pave a major road to the development of mitochondrial dysfunction and impaired oxidative metabolism seen in many patients diagnosed with these disorders.
... Finally, a good deal has been written about the interaction of peroxynitrite with cytochrome c. [38,[149][150][151][152]. Radi and co-workers first described the interaction to be a direct one which did not involve any intermediates. ...
... The loss of function was demonstrated, for example, on MnSOD (manganese superoxide dismutase, a mitochondrial enzyme) [72] or PGI 2 (prostacyclin (prostaglandin I 2 ) synthase, a vascular enzyme) [73]. The gain of function was demonstrated, for example, on cytochrome c, which gains peroxidase activity [74,75]; on fibrinogen (higher aggregation in coagulation) [76]; or on protein kinase C [77] (summary in Table 3, adapted from [71]). ...
Aging is a complex process of organism decline in physiological functions. There is no clear theory explaining this phenomenon, but the most accepted one is the oxidative stress theory of aging. Biomarkers of oxidative stress, substances, which are formed during oxidative damage of phospholipids, proteins, and nucleic acids, are present in body fluids of diseased people as well as the healthy ones (in a physiological concentration). 8-
iso
prostaglandin F
2
α
is the most prominent biomarker of phospholipid oxidative damage,
o
-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine are biomarkers of protein oxidative damage, and 8-hydroxy-2′-deoxyguanosine and 8-hydroxyguanosine are biomarkers of oxidative damage of nucleic acids. It is thought that the concentration of biomarkers increases as the age of people increases. However, the concentration of biomarkers in body fluids is very low and, therefore, it is necessary to use a sensitive analytical method. A combination of HPLC and MS was chosen to determine biomarker concentration in three groups of healthy people of a different age (twenty, forty, and sixty years) in order to find a difference among the groups.
... Peroxynitrite may additionally inactivate Mn superoxide dismutase further amplifying mitochondrial injury. Peroxynitrite also targets cytochrome c resulting in elevated peroxidase activity which further aggravates the peroxynitriteinduced damage to proteins and membranes (Jang and Han 2006;Batthyany et al. 2005). The consequent ROS production may cause mitochondrial dysfunctions and consequent apoptosis (Lo et al. 2010;Smith and Murphy 2010). ...
Myalgic encephalomyelitis / chronic fatigue syndrome (ME/cfs) is classified by the World Health Organization as a disorder of the central nervous system. ME/cfs is an neuro-immune disorder accompanied by chronic low-grade inflammation, increased levels of oxidative and nitrosative stress (O&NS), O&NS-mediated damage to fatty acids, DNA and proteins, autoimmune reactions directed against neoantigens and brain disorders. Mitochondrial dysfunctions have been found in ME/cfs, e.g. lowered ATP production, impaired oxidative phosphorylation and mitochondrial damage. This paper reviews the pathways that may explain mitochondrial dysfunctions in ME/cfs. Increased levels of pro-inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-α, and elastase, and increased O&NS may inhibit mitochondrial respiration, decrease the activities of the electron transport chain and mitochondrial membrane potential, increase mitochondrial membrane permeability, interfere with ATP production and cause mitochondrial shutdown. The activated O&NS pathways may additionally lead to damage of mitochondrial DNA and membranes thus decreasing membrane fluidity. Lowered levels of antioxidants, zinc and coenzyme Q10, and ω3 polyunsaturated fatty acids in ME/cfs may further aggravate the activated immuno-inflammatory and O&NS pathways. Therefore, it may be concluded that immuno-inflammatory and O&NS pathways may play a role in the mitochondrial dysfunctions and consequently the bioenergetic abnormalities seen in patients with ME/cfs. Defects in ATP production and the electron transport complex, in turn, are associated with an elevated production of superoxide and hydrogen peroxide in mitochondria creating adaptive and synergistic damage. It is argued that mitochondrial dysfunctions, e.g. lowered ATP production, may play a role in the onset of ME/cfs symptoms, e.g. fatigue and post exertional malaise, and may explain in part the central metabolic abnormalities observed in ME/cfs, e.g. glucose hypometabolism and cerebral hypoperfusion.
... The rate constant of this reaction is not known, yet a k value comparable to or slightly smaller than that for the reaction of NO 2 d with TyrO d seems reasonable. In the case of cytochrome c, formation of nitrotryptophan has been observed at high peroxynitrite concentration (4 4 mM) [74]. Here, we have assumed that Trp d reacts with NO 2 d as fast as TyrO d (Reaction (11)), and the k 9 value has been derived under this assumption. ...
The reactions of NO2(•) with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyl-tyrosine amide and N-acetyl-tryptophan amide at pH 7.3 were studied by pulse radiolysis at 23°C. NO2(•) oxidizes N-acetyl-tyrosine amide and N-acetyl-tryptophan amide with rate constants of (3.1±0.3)×10(5) and (1.1±0.1)×10(6)M(-1)s(-1), respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10(6)M(-1)s(-1) at pH 7.2. NO2(•) oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10(7)M(-1)s(-1) at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2(•) oxidizes the iron center directly - most probably via reaction at the solvent-accessible haem-edge - whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2(•) will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.
... Mechanism of bioactivation by other heme proteins: Cytochrome c, cytoglobin, cytochrome c oxidase, and endothelial NOS-Similar to Ngb, the heme iron of cytochrome c is predominately hexacoordinate, but can become pentacoordinate when the iron-methionine bond is ruptured which occurs when the methionine is oxidized [65,66], tyrosines are nitrated [67,68], or the cytochrome interacts with anionic phospholipids such as those present in the inner membrane of the mitochondria [69][70][71]. Zweier, Mason and colleagues demonstrated cytochrome-c mediated nitrite reduction secondary to protein oxidation by hypochlorite [72]. ...
It is now accepted that the anion nitrite, once considered an inert oxidation product of nitric oxide (NO), contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling. As such, its application in therapeutics is being actively testing in pre-clinical models and in human phase I-II clinical trials. Major pathways for nitrite bioactivation involve its reduction to NO by members of the hemoglobin or molybdopterin family of proteins, or catalyzed dysproportionation. These conversions occur preferentially under hypoxic and acidic conditions. A number of enzymatic systems reduce nitrite to NO and their activity and importance are defined by oxygen tension, specific organ system and allosteric and redox effectors. In this work, we review different proposed mechanisms of nitrite bioactivation, focusing on analysis of kinetics and experimental evidence for the relevance of each mechanism under different conditions.
Mechanical perturbation triggers activation of resident myogenic stem cells to enter the cell cycle through a cascade of events including hepatocyte growth factor (HGF) release from its extracellular tethering and the subsequent presentation to signaling‐receptor c‐met. Here, we show that with aging, extracellular HGF undergoes tyrosine‐residue (Y) nitration and loses c‐met binding, thereby disturbing muscle homeostasis. Biochemical studies demonstrated that nitration/dysfunction is specific to HGF among other major growth factors and is characterized by its locations at Y198 and Y250 in c‐met‐binding domains. Direct‐immunofluorescence microscopy of lower hind limb muscles from three age groups of rat, provided direct in vivo evidence for age‐related increases in nitration of ECM‐bound HGF, preferentially stained for anti‐nitrated Y198 and Y250‐HGF mAbs (raised in‐house) in fast IIa and IIx myofibers. Overall, findings highlight inhibitory impacts of HGF nitration on myogenic stem cell dynamics, pioneering a cogent discussion for better understanding age‐related muscle atrophy and impaired regeneration with fibrosis (including sarcopenia and frailty).
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the production of renal ischaemia/reperfusion (I/R). The current study is to elucidate a mechanism of SIRT2 tyrosine nitration to accelerate the cell apoptosis induced by peroxynitrite (ONOO‾), the most reactive and deleterious RNS type in renal ischaemia/reperfusion (I/R) injury. Our results demonstrate that there is a significant enhancement of the 3-nitrotyrosine levels in renal tissues of Acute Kidney Injury (AKI) patients and rats that underwent renal I/R, and a positive correlation between the 3-nitrotyrosine level and renal function impairment, indicative of an accumulation of peroxynitrite. Notably, peroxynitrite-evoked nitration of SIRT2 destroyed its enzymatic activity and the capability to deacetylate FOXO3a, and enhanced expression of Bim and caspase3, facilitating renal cell apoptosis in renal ischaemia/reperfusion and SIN-1(peroxynitrite donor) treatment in vitro, and these effects were reversed by FeTMPyP, a peroxynitrite decomposition scavenger. Importantly, we identified that the tyrosine 86 is responsible for SIRT2 nitration and inactivation using site-mutation assay and Mass Spectrography analysis. Altogether, these findings point to a novel protective mechanism that an inhibition of SIRT2 tyrosine nitration can be a promising strategy to prevent ischaemic renal diseases involving AKI.
Cytochrome c (Cyt c) is a multifunctional protein that, in its native conformation, shuttles electrons in the mitochondrial respiratory chain. Conformational transitions that involve replacement of the heme distal ligand lead to the gain of alternative peroxidase activity, which is crucial for membrane permeabilization during apoptosis. Using a time-resolved SERR spectroelectrochemical approach, we found that the key physicochemical parameters that characterize the electron transfer (ET) canonic function and those that determine the transition to alternative conformations are strongly correlated and are modulated by local electric fields (LEF) of biologically meaningful magnitude. The electron shuttling function is optimized at moderate LEFs of around 1 V nm⁻¹. A decrease of the LEF is detrimental for ET as it rises the reorganization energy. Moreover, LEF values below and above the optimal for ET favor alternative conformations with peroxidase activity and downshifted reduction potentials. The underlying proposed mechanism is the LEF modulation of the flexibility of crucial protein segments, which produces a differential effect on the kinetic ET and conformational parameters of Cyt c. These findings might be related to variations in the mitochondrial membrane potential during apoptosis, as the basis for the switch between canonic and alternative functions of Cyt c. Moreover, they highlight the possible role of variable LEFs in determining the function of other moonlighting proteins through modulation of the protein dynamics.
Glutamine synthetase (GS) catalyzes an ATP-dependent condensation of glutamate and ammonia to form glutamine. This reaction – and therefore GS – are indispensable for the hepatic nitrogen metabolism. Nitration of tyrosine 336 (Y336) inhibits human GS activity. GS nitration and the consequent loss of GS function are associated with a broad range of neurological diseases. The mechanism by which Y336 nitration inhibits GS, however, is not understood. Here, we show by means of unbiased MD simulations, binding and configurational free energy computations that Y336 nitration hampers ATP binding, but only in the deprotonated and negatively-charged state of residue 336. By contrast, for the protonated and neutral state, our computations indicate an increased binding affinity for ATP. pKa computations of nitrated Y336 within GS predict a pKa of ~5.3. Thus, at physiological pH nitrated Y336 exists almost exclusively in the deprotonated and negatively-charged state. In vitro experiments confirm these predictions, in that, the catalytic activity of nitrated GS is decreased at pH 7 and pH 6, but not at pH 4. These results indicate a novel, fully reversible, pH-sensitive mechanism for the regulation of GS activity by tyrosine nitration.
Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and that is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-Hand metal-binding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. NMR spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-Hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. In order to develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF Hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity (Kd = 1.6 µM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched on nitration. The greater terbium affinity upon tyrosine nitration resulted in high dynamic range and sensitivity to sub-stoichiometric nitration. An improved approach was also developed to the synthesis of peptides containing nitrotyrosine, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.
In this review, I provide historical background on how nitric oxide ( • NO) and peroxynitrite research was originated in Uruguay in the early 90′s and how the investigations evolved through over more than two decades within a context related to human biology. This process involved the participation of multiple local investigators, in conjunction with collaborations at the regional (Latin American) and international levels. The discoveries have been integrated with parallel investigations from other research groups worldwide and, have provided a body of knowledge to unravel how the free radical nitric oxide ( • NO) can shift its signal transduction action towards oxidative processes via its interactions with superoxide radical (O 2•– ) to yield peroxynitrite, a strong biological oxidant. The oxidative biochemistry of peroxynitrite involves both direct reactions and the formation of secondary oxidizing species (i.e. hydroxyl radicals ( • OH), carbonate radicals (CO 3•– ) and nitrogen dioxide ( • NO 2 )) that cause oxidative modifications of biomolecules, including thiol oxidation and tyrosine nitration. Due to the intrinsic instability of peroxynitrite in biological systems, its half-life and fate are largely dictated by its reaction kinetics with biotargets. The direct actions of • NO and peroxynitrite in the modulation of intracellular redox processes are disparate, with peroxynitrite typically causing permanent modifications of cellular components and resulting in severe alterations of cell and mitochondrial homeostasis. Herein, I highlight the evolution and progression of • NO and peroxynitrite research in Uruguay during over 25 years of work, emphasizing hypothesis- and mechanistic-oriented biochemical studies and their translation to medically-relevant conditions.
Protein tyrosine (Tyr) nitration, the covalent addition of a nitro group (•NO2) to Tyr residues, is emerging as a candidate mechanism of endothelial dysfunction. Previous studies have shown that Tyr nitration is primarily induced by nitrosative stress, a process characterized by the production of reactive nitrogen species, especially peroxynitrite anion (ONOO⁻), which is considered a secondary product of NO in the presence of superoxide radicals (O2•−). However, the impact of nitrosative stress–induced Tyr nitration on endothelial dysfunction has not been thoroughly elucidated to date. We developed an endothelial dysfunction model, a process called “endothelial‐to‐mesenchymal transition (EndMT),” and evaluated the production of NO, O2•−, and protein nitration during EndMT. The results showed that TGF‐β1 stimulation induced EndMT and elevated endothelial NO and O2•− production as well as nitration of the catalytic subunit of protein phosphatase (PP)2A. Mass spectrometry analysis showed that Tyr265 was the nitration site in the catalytic subunit of protein phosphatase (PP)2A, and this Tyr nitration increased PP2A activity and disrupted endothelial integrity. To devise an endothelial‐targeted anti‐PP2Ac nitration strategy, a mimic peptide, tyrosine 265 wild type (Y265WT), conjugated with the cell‐penetrating peptide HIV‐1 TAT protein (TAT) was synthesized. PP2Ac nitration and PP2A activity were significantly inhibited by pretreatment with TAT‐265WT, and the integrity of endothelial cells was maintained. Furthermore, injection of TAT‐265WT attenuated renal nitration formation and caused anticapillary rarefaction in a unilateral urethral obstructive nephropathy model. Taken together, these results offer preclinical proof of concept for TAT‐265WT as a tractable agent to protect against nitrosative stress‐induced endothelial dysfunction in renal microvessels.—Deng, Y., Cai, Y., Liu, L., Lin, X., Lu, P., Guo, Y., Han, M., Xu, G. Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress–induced endothelial dysfunction in renal microvessels. FASEB J. 33, 3718–3730 (2019). www.fasebj.org
Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•–) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.
Peroxynitrite (–OON=O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative (bio)chemistry. It may derive from reaction of superoxide anion (O2•–) with nitric oxide (•NO) and has been suggested to form an as-yet unobserved bound heme-iron-PN intermediate in the catalytic cycle of Nitric Oxide Dioxygenase (NOD) enzymes, which facilitate a •NO homeostatic process, i.e., its oxidation to the nitrate anion. Here, a discrete six-coordinate low-spin porphyrinate-FeIII complex [(PIm)FeIII(–OON=O)] (3) (PIm; a porphyrin moiety with a covalently tethered imidazole axial ‘base’ donor ligand) has been identified and characterized by various spectroscopies (UV-Vis, NMR, EPR, XAS, resonance Raman) and DFT calculations, following its formation at –80 °C by addition of •NO(g) to the heme-superoxo species, [(PIm)FeIII(O2•-)] (2). DFT calculations confirm that 3 is a six-coordinate low-spin species with the PN ligand coordinated to iron via its terminal peroxidic anionic O-atom with the overall geometry being in a cis-configuration. Complex 3 thermally transforms to its isomeric low-spin nitrato form [(PIm)FeIII(NO3-)] (4a). While previous (bio)chemical studies show that phenolic substrates undergo nitration in the presence of PN or PN-metal complexes, in the present system, addition of 2,4-di-tert-butylphenol (2,4DTBP) to complex 3 does not lead to nitrated phenol; the nitrate complex 4a still forms. DFT calculations reveal that the phenolic H-atom approaches the terminal PN O-atom (farthest from the metal center and ring core), effecting O–O cleavage, giving nitrogen dioxide (•NO2) plus a ferryl compound [(PIm)FeIV=O] (7); this rebounds to give [(PIm)FeIII(NO3-)] (4a).The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished.
Mitochondria are integral to both the maintenance of cellular homeostasis and the regulation of cell death. Mitochondrial function is highly dynamic and tightly regulated, and the reactive and ubiquitous nature of NO as a signaling molecule led to its early identification as a potential regulator of mitochondrial function on a number of levels. It is now recognized that NO interacts with the mitochondrion at multiple sites and through several different molecular mechanisms, including modulating mitochondrial structure and number, regulating basic organellar functions from bioenergetics to signaling, and converse regulation of NO itself by the unique environment found in the organelle. While the 1990s and early 2000s focused on elucidating the biochemical mechanisms of NO–mitochondrial interactions, the relevance of these interactions for various disease states is still being elucidated. In this chapter we will both summarize the field to date and catalog some areas in need of further investigation.
Over the past several decades, nanotechnology has contributed to the progress of biomedicine, biomarker discovery, and the development of highly sensitive electroanalytical / electrochemical biosensors for in vitro and in vivo monitoring, and quantification of oxidative and nitrosative stress markers like reactive oxygen species (ROS) and reactive nitrogen species (RNS). A major source of ROS and RNS is oxidative stress in cells, which can cause many human diseases, including cancer. Therefore, the detection of local concentrations of ROS (e. g. superoxide anion radical; O2(•-) ) and RNS (e. g. nitric oxide radical; NO(•) and its metabolites) released from biological systems is increasingly important and needs a sophisticated detection strategy to monitor ROS and RNS in vitro and in vivo. In this review, we discuss the nanomaterials-based ROS and RNS biosensors utilizing electrochemical techniques with emphasis on their biomedical applications.
Significance:
"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function.
Critical issues:
Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation.
Future directions:
The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.
Protein tyrosine nitration is an important posttranslational modification involving a variety of diseases. It's occurred via peroxynitrite or nitrite/hydrogen peroxide/hemeperoxydase system, and nitrotyrosine is formed by free radical reaction. The in vivo protein nitration pathways, the mechanism and the biological significance are discussed. It points out that protein nitration has selectivity, and nitration of special tyrosine residue(s) can lead to the alteration of the structure and functions of the protein, and affect the immunological response or signal transduction involved.
Peroxynitrite, the product of the fast reaction between nitric oxide and superoxide radicals, is a strong oxidant and cytotoxic agent in vivo. It can attack biological molecules, including proteins, lipids and DNA. In recent years, the interactions of peroxynitrite and proteins have attracted considerable interest because peroxynitrite-mediated protein damage has been found in a variety of diseases. In this paper, we discuss the potential pathological significance of protein damage induced by peroxynitrite in detail, and the molecular mechanisms of protein modification by peroxynitrite are reviewed as well. Moreover, the future developments of the interaction between peroxynitrite and protein are also prospected.
This paper presented at the European Meeting of the Society-for-Free-Radical-Research-Europe 2007, discusses the development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease.
Protein nitration is an important post-translational modification regulating protein structure and function, especially for heme proteins. Myoglobin (Mb) is an ideal protein model for investigating the structure and function relationship of heme proteins. With limited structural information available for nitrated heme proteins from experiments, we herein performed a molecular dynamics study of human Mb with successive nitration of Tyr103, Tyr146, Trp7 and Trp14. We made a detailed comparison of protein motions, intramolecular contacts and internal cavities of nitrated Mbs with that of native Mb. It showed that although nitration of both Tyr103 and Tyr146 slightly alters the local conformation of heme active site, further nitration of both Trp7 and Trp14 shifts helix A apart from the rest of protein, which results in altered internal cavities and forms a water channel, representing an initial stage of Mb unfolding. The computational study provides an insight into the nitration of heme proteins at an atomic level, which is valuable for understanding the structure and function relationship of heme proteins in non-native states by nitration.
The effect of addition of the nitric oxide donor S-nitrosoglutathione (GSNO) on the Zn nutritional status was evaluated in hydroponically-cultured wheat plants (Triticum aestivum cv. Chinese Spring). Addition of GSNO in Zn-deprived plants did not modify biomass accumulation but accelerated leaf senescence in a mode concomitant with accelerated decrease of Zn allocation to shoots. In well-supplied plants, Zn concentration in both roots and shoots declined due to long term exposure to GSNO. A further evaluation of net Zn uptake rate (ZnNUR) during the recovery of long-term Zn-deprivation unveiled that enhanced Zn-accumulation was partially blocked when GSNO was present in the uptake medium. This effect on uptake was mainly associated with a change of Zn translocation to shoots. Our results suggest a role for GSNO in the modulation of Zn uptake and in root-to-shoot translocation during the transition from deficient to sufficient levels of Zn-supply.
Peroxynitrite (ONOO-), which is usually generated as a response of the immune system or in inflammatory processes, is a powerful oxidant species, while hemoglobin is an important peroxynitrite scavenger in vivo. In this work, we have studied the interaction between peroxynitrite and hemoglobin through an electrochemical method and UV-Vis spectroscopy. It is found that peroxynitrite, at a relatively high concentration level, may make the protein exhibit a concentration-dependent increase of its catalytic activity towards hydrogen peroxide, whereas peroxynitrite at low concentration will result in the slight decrease of the catalytic activity. Further studies reveal that the diversification of the enzymatic activity is ascribed to the different extent of tyrosine nitration and, accordingly, the spatial conformation of the protein.
Nitrite is one of the players in the broad nitrogen biogeochemical cycle. This nitrogen oxo-anion is involved in key pathways crucial to life on Earth and to the planetary recycling of nitrogen. The global aim is to review the present functional, structural, and mechanistic knowledge of nitrite reduction/oxidation, to assess in what extent we understand how nitrite is handled by living organisms. Nitrite formation is outside the scope of this Review. This knowledge is essential for the comprehension of the global nitrogen biochemical cycle and, consequently, for the comprehension of the impressive changes the human activities are introducing in the cycle. However, under most physiological conditions, glutamate dehydrogenase catalyzes instead the reverse reaction, yielding ammonium in amino acid catabolism.
Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-β (TGF-β) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-β-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.
Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas
cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine
72,N-ε-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome cexpressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and
other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic
activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.
Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.
The tyrosine-67 to phenylalanine mutated rat cytochrome c is similar to the unmutated protein in its spectral, reduction potential, and enzymic electron-transfer properties. However, the loss of the 695-nm band, characteristic of the ferric form of the normal low-spin physiologically active configuration, occurs 1.2 pH units higher on the alkaline side and 0.7 pH unit lower on the acid side. Similarly, the heme iron-methionine-80 sulfur bond is more stable to temperature, with the midpoint of the transition being 30 degrees C higher, corresponding to an increase in delta H of 5 kcal/mol (1 cal = 4.184 J), partially mitigated by an increase of 11 entropy units in delta S. Urea has only slightly different effects on the two proteins. These phenomena are best explained by considering that the loss of one of the three hydrogen-bonding side chains, tyrosine-67, asparagine-52, and threonine-78, which hold an internal water molecule on the "left, lower front" side of the protein [Takano, T. & Dickerson, R. E. (1981) J. Mol. Biol. 153, 95-115], is sufficient to prevent its inclusion in the mutant protein, leading to a more stable structure, and, as indicated by preliminary proton NMR two-dimensional phase-sensitive nuclear Overhauser effect spectroscopy analyses, a reorganization of this area. This hypothesis predicts that elimination of the hydrogen-bonding ability of residue 52 or 78 would also result in cytochromes c having similar properties. It is not obvious why the space-filling structure involving the internalized water molecule that leads to a destabilization energy of about 3 kcal/mol should be subject to extreme evolutionary conservation, when a more stable and apparently fully functional structure is readily available.
Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.
Nitric oxide (.NO) plays a central role in the pathogenesis of diverse inflammatory and infectious disorders. The toxicity of .NO is thought to be engendered, in part, by its reaction with superoxide (O2.-), yielding the potent oxidant peroxynitrite (ONOO-). However, evidence for a role of ONOO- in vivo is based largely upon detection of 3-nitrotyrosine in injured tissues. We have recently demonstrated that nitrite (NO2-), a major end-product of .NO metabolism, readily promotes tyrosine nitration through formation of nitryl chloride (NO2Cl) and nitrogen dioxide (.NO2) by reaction with the inflammatory mediators hypochlorous acid (HOCl) or myeloperoxidase. We now show that activated human polymorphonuclear neutrophils convert NO2- into NO2Cl and .NO2 through myeloperoxidase-dependent pathways. Polymorphonuclear neutrophil-mediated nitration and chlorination of tyrosine residues or 4-hydroxyphenylacetic acid is enhanced by addition of NO2- or by fluxes of .NO. Addition of 15NO2- led to 15N enrichment of nitrated phenolic substrates, confirming its role in polymorphonuclear neutrophil-mediated nitration reactions. Polymorphonuclear neutrophil-mediated inactivation of endothelial cell angiotensin-converting enzyme was exacerbated by NO2-, illustrating the physiological significance of these reaction pathways to cellular dysfunction. Our data reveal that NO2- may regulate inflammatory processes through oxidative mechanisms, perhaps by contributing to the tyrosine nitration and chlorination observed in vivo.
Cyanide (CN(-)) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a(3) of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ((.)CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(.)CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N(3)(-)). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/(.)N(3) radical adduct was readily detected when H(2)O(2) was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.
Peroxynitrite (ONOO(-)), the product of superoxide (O(2)) and nitric oxide (.NO) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO(-) during conditions involving accelerated rates of oxygen radical and.NO generation. Horse heart cytochrome c(3+) was nitrated by ONOO(-), as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c(3+) 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO(2)) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochrome c(3+) was more extensively nitrated than cytochrome c(2+) because of the preferential oxidation of the reduced heme by ONOO(-). Similar protein nitration patterns were obtained by ONOO(-) reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO(2)(-)) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during .NO- and ONOO(-)-mediated cell injury.
Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochrome c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300-600 and 200-320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe(3+). The studies also include the effect of P(i), NaCl, and CaCl(2). Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome c with charged interfaces promotes conformational changes in the alpha-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochrome c. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl(2), NaCl, and P(i), in response to the increase of the ionic strength.
Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased approximately 50% in angiotensin II-infused rat kidneys (24 +/- 4.6 vs. 11 +/- 5.2 U/mg) without a change in protein expression. Immunohistochemical staining showed 3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine colocalized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney [3.2 +/- 1.9 (sham treated) vs. 9.5 +/- 2.3 ng/mg protein by HPLC-UV detection]. With tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of 3-nitrotyrosine by Western blotting or HPLC. By HPLC, 3-nitrotyrosine content of kidney MnSOD increased 13-fold after angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total 3-nitrotyrosine content in sham-treated and angiotensin II-infused rat kidney, respectively. Angiotensin II-induced hypertension is accompanied by increased tyrosine nitration of MnSOD, which, because it inactivates the enzyme, may contribute to increased oxidant stress in the kidney.
Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-E(K)-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC(88-103)) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC(88-103) with nitrotyrosine abrogates recognition by the MCC(88-103)-specific T cell hybridoma 2B4. CBA (H2(K)) mice immunized with MCC(88-103) or nitrated MCC(88-103) peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC(88-103), but exhibited a robust immune response against nitrated MCC(88-103). Analysis of T cell hybridomas specific for nitrated-MCC(88-103) indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.
Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H2O2 revealed a single-line signal with a line width of ∼10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn2+, indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.-). The production of cyt c-derived ToQ.- is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H 2O2 as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H2O2 was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H2O2 revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H 2O2, NO, and apoptosis.
Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G' value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.
This chapter provides a comprehensive overview of the physical and biological chemistry of peroxynitrite. A foundation is provided to rationalize the biological fate and actions of peroxynitrite and the strategies for preventing peroxynitrite-dependent biological damage and pathology. Peroxynitrite anion is formed in vivo as a result of the diffusion controlled reaction between nitric oxide (NO) and superoxide anion radicals. The anion and its conjugated acid, peroxynitrous acid, are strong oxidant species that cause molecular damage in a variety of pathophysiological conditions. Peroxynitrite reacts fast with a number of biological targets, including thiols, metalloproteins, and carbon dioxide, or more slowly decomposes to hydroxyl and nitrogen dioxide radicals by proton-catalyzed homolysis. Carbon dioxide accounts for a significant fraction of peroxynitrite consumption and leads to the secondary formation of carbonate and nitrogen dioxide radicals. At the molecular level, the predominant outcome of peroxynitrite reactions in vivo is one or two electron oxidations and nitrations. Peroxynitrite can diffuse through tissue compartments, being able to cross biomembranes by both passive diffusion and anion channels. Thus, although the biological half-life of peroxynitrite is short, it is sufficient for peroxynitrite to diffuse a couple of cell diameters and cause biological effects distant from its site of production.
The term cytochrome c is both a spectral and a structural classification, related more to the heme and its attachment to the polypeptide chain than to the protein which surrounds it. The most familiar member of the class is cytochrome c from the mitochondria1 respiratory chain, which has a single heme group per chain of 103–113 amino acids, and a reduction potential of +260 mV. Cytochromes all have a characteristic three-banded absorption spectrum in the reduced state. One of the most striking features of the heme group is the delocalization of electrons among the π orbitals of the porphyrin ring. The Soret band in the absorption spectrum represents the excitation of delocalized π electrons to unoccupied levels of the porphyrin ring of similar angular momentum. The absorption spectrum of cytochrome c, which is the basis for classification, depends on the side groups around the porphyrin ring and the way that they are connected to the protein chain. All cytochromes c are oxidation-reduction proteins involved in either respiration or photosynthesis.
Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.
Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.
Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.
Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
A series of radical transitions, Br2-.----Met(S therefore Br)----Trp(indolyl)----Tyr (phenoxyl), has been demonstrated by pulse radiolysis of N2O-saturated aqueous solutions containing Br-, Met-Gly and Trp-(Gly)2-Tyr at pH 6.7. The intramolecular Met(S therefore Br)----Trp(indolyl) transition in the dipeptide Met-Trp is shown to proceed via the Trp+. radical cation, with a rate constant of k approximately 10(7)s-1, consistent with an electron transfer. Br2-.-attack upon ribonuclease A (RNase) leads to a fast Met(S therefore Br)----Tyr(phenoxyl) process, k = (4.0 +/- 1.0) X 10(5)s-1, probably involving the solvent-exposed Met-29 and the adjacent Tyr-25. Phenoxyl dimerization in the RNase system produces the characteristic o,o'-biphenol fluorescence, but a competing interaction of the Tyr-25(phenoxyl) with the 26-84 disulphide group also appears possible.
Cytochrome c3+ has been extensively used for the detection of superoxide produced in biological systems due to its fast superoxide-mediated reduction to cytochrome c2+. However, another biomolecule which is sometimes cogenerated with superoxide, nitric oxide, reacts with superoxide at almost diffusion-controlled rates (6.7 x 10(9) M-1 s-1), leading to the production of a highly oxidizing species, peroxynitrite anion (ONOO-). In this work we report that peroxynitrite readily oxidizes cytochrome c2+ to cytochrome c3+ in an ascorbate-reversible manner. The reaction between peroxynitrite and cytochrome c2+ occurs with a second-order rate constant of 2.3 x 10(5) M-1 s-1. The pH dependence of the apparent second-order rate constants as well as the effect of different scavengers indicated that peroxynitrous acid (ONOOH) in the ground state was the actual species responsible of cytochrome c2+ oxidation. The activation enthalpy, free energy, and entropy were +10.8 kcal mol-1, +11.8 kcal mol-1, and -3.15 cal mol-1 K-1, respectively, in agreement with the proposed reaction mechanism. Additionally, our results imply that when quantitating superoxide by the cytochrome c3+ reduction method, the existence of a simultaneous generation of nitric oxide and peroxynitrite may lead to an underestimation of the rates of superoxide production.
Peroxynitrite (ONOO-), the reaction product of superoxide (O2.-) and nitric oxide (.NO), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. At high peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from peroxynitrite increased at acidic pH, with an apparent pKa of 7.8. High concentrations of Fe(3+)-EDTA were required to enhance peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 microM Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration. Plots of the observed rate constant of nitrotryptophan formation vs tryptophan concentration presented downward curvatures. Thus, the kinetics of metal-independent nitration reactions were interpreted in terms of two parallel mechanisms. In the first one, ground-state peroxynitrous acid nitrated tryptophan with a second-order rate constant of 184 +/- 11 M-1 s-1 at 37 degrees C. The activation enthalpy was 9.1 +/- 0.3 kcal mol-1, and the activation entropy was -19 +/- 1 cal mol-1 K-1. In the second mechanism, ONOOH*, an activated intermediate derived from trans-peroxynitrous acid formed in a steady state, was the nitrating agent.
Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3). Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3. Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1. Both proteins were expressed in the cytoplasm of E. coli cells harboring the plasmid. Semianaerobic cultures grown in a fermentor produced 15 mg of recombinant iso-1-cytochrome c per liter of culture. Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population. Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E. coli was compared to the same protein expressed in yeast. At neutral pH, the two proteins exhibit indistinguishable spectroscopic and physical (Tm, Em') characteristics. However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E. coli as it is by S. cerevisiae. Interestingly, the pKa of the alkaline transition of the protein expressed in E. coli is approximately 0.6 pKa unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7). 1H NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast. These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine.
Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.
Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.
Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.
Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.
Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.
Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.
Peroxynitrite promotes oxidative damage and is implicated in the pathophysiology of various diseases that involve accelerated rates of nitric oxide and superoxide formation. The unambiguous detection of peroxynitrite in biological systems is, however, difficult due to the combination of a short biological half-life, limited diffusion, multiple target molecule reactions, and participation of alternative oxidation/nitration pathways. In this review, we provide the conceptual framework and a comprehensive analysis of the current experimental strategies that can serve to unequivocally define the existence and quantitation of peroxynitrite in biological systems of different levels of organization and complexity.
Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO
2−). Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3-nitrotyrosine. In this work, we show that mass spectral patterns associated with 3-nitrotyrosine containing peptides allow identification of peptides containing this modification. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to characterize a synthetic peptide AAFGY(m-NO2)AR and several peptides containing 3-nitrotyrosine derived from bovine serum albumin treated with tetranitromethane. A unique series of ions were found for these peptides in addition to the mass shift of +45 Da corresponding to the addition of the nitro group. Specifically, two additional ions were observed at roughly equal abundance that correspond to the loss of one and two oxygens, and at lower abundances, two ions are seen that suggest the formation of hydroxylamine and amine derivatives. These latter four components appear to originate by laser-induced photochemical decomposition. MALDI-MS analysis of the synthetic peptide containing 3-nitrotyrosine revealed this same pattern. Post-source decay (PSD) MALDI-time-of-flight (TOF) and collisional activation using a prototype MALDI quadrupole TOF yielded extensive fragmentation that allowed site-specific identification of 3-nitrotyrosine. Conversion of peptides containing 3-nitrotyrosine to 3-aminotyrosine with Na2S2O4 yielded a single molecular ion by MALDI with an abundant sidechain loss under PSD conditions. These observations suggest that MALDI can provide a selective method for the analysis and characterization of 3-nitrotyrosine-containing peptides.
In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.
Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.
Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2-52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.
Mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, and these interactions are recognized to contribute to the biological and pathological effects of both nitric oxide ((*)NO) and peroxynitrite. Extra- or intramitochondrially formed peroxynitrite can diffuse through mitochondrial compartments and undergo fast direct and free radical-dependent target molecule reactions. These processes result in oxidation, nitration, and nitrosation of critical components in the matrix, inner and outer membrane, and intermembrane space. Mitochondrial scavenging and repair systems for peroxynitrite-dependent oxidative modifications operate but they can be overwhelmed under enhanced cellular (*)NO formation as well as under conditions that lead to augmented superoxide formation by the electron transport chain. Peroxynitrite can lead to alterations in mitochondrial energy and calcium homeostasis and promote the opening of the permeability transition pore. The effects of peroxynitrite in mitochondrial physiology can be largely rationalized based on the reactivities of peroxynitrite and peroxynitrite-derived carbonate, nitrogen dioxide, and hydroxyl radicals with critical protein amino acids and transition metal centers of key mitochondrial proteins. In this review we analyze (i) the existing evidence for the intramitochondrial formation and reactions of peroxynitrite, (ii) the key reactions and fate of peroxynitrite in mitochondria, and (iii) their impact in mitochondrial physiology and signaling of cell death.
Apoptosis, or programmed cell death, is involved in development, elimination of damaged cells, and maintenance of cell homeostasis. Deregulation of apoptosis may cause diseases, such as cancers, immune diseases, and neurodegenerative disorders. Apoptosis is executed by a subfamily of cysteine proteases known as caspases. In mammalian cells, a major caspase activation pathway is the cytochrome c-initiated pathway. In this pathway, a variety of apoptotic stimuli cause cytochrome c release from mitochondria, which in turn induces a series of biochemical reactions that result in caspase activation and subsequent cell death. In this review, we focus on the recent progress in understanding the biochemical mechanisms and regulation of the pathway, the roles of the pathway in physiology and disease, and their potential therapeutic values.
Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.
The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the signal transducing physiological actions of (.)NO to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite anion (ONOO(-)) and nitrogen dioxide ((.)NO2), formed as secondary products of (.)NO metabolism in the presence of oxidants including superoxide radicals (O2(.-)), hydrogen peroxide (H2O2), and transition metal centers. The precise interplay between (.)NO and oxidants and the identification of the proximal intermediate(s) responsible for nitration in vivo have been under controversy. Despite the capacity of peroxynitrite to mediate tyrosine nitration in vitro, its role on nitration in vivo has been questioned, and alternative pathways, including the nitrite/H2O2/hemeperoxidase and transition metal-dependent mechanisms, have been proposed. A balanced analysis of existing evidence indicates that (i) different nitration pathways can contribute to tyrosine nitration in vivo, and (ii) most, if not all, nitration pathways involve free radical biochemistry with carbonate radicals (CO3(.-)) and/or oxo-metal complexes oxidizing tyrosine to tyrosyl radical followed by the diffusion-controlled reaction with (.)NO2 to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine has been revealed as a relevant biomarker of (.)NO-dependent oxidative stress; additionally, site-specific nitration focused on particular protein tyrosines may result in modification of function and promote a biological effect. Tissue distribution and quantitation of protein 3-nitrotyrosine, recognition of the predominant nitration pathways and individual identification of nitrated proteins in disease states open new avenues for the understanding and treatment of human pathologies.