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The nature of heme/iron-induced protein tyrosine nitration
Abstract
Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H(2)O(2) to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO(2)-Tyr formation. The data from our study indicate that: (i) hemeiron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H(2)O(2), and NO(2)(-); (ii) H(2)O(2) plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H(2)O(2), and the NO(2)(-) system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O(2)(*-) (ONOO(-) formation), (*)OH (Fenton reaction), and compound III are unlikely to contribute to the reaction; and (iv) hemoprotein-rich tissues such as cardiac muscle are vulnerable to protein nitration in pathological conditions characterized by the overproduction of H(2)O(2) and NO(2)(-), or nitric oxide.
... For instance, peroxynitrite (ONOO •-) has been considered one of the main nitrating species for proteins [10]. In addition, protein nitration induced by nitrite (NO 2 − ) and hydrogen peroxide (H 2 O 2 ), as the stable end products of • NO and oxygen free radicals, is another mechanism with the potential catalytic effects of hemin [11,12]. Furthermore, through its nitrosylating effects for the tyrosine residues within the protein structure, • NO was found to provide a mechanism for further protein nitration [13]. ...
... In the presence of hemin, the general mechanisms by which it can induce the nitration of cellular proteins have been reported before. In these mechanisms, NO 2 − , as the stable end product of • NO metabolism and H 2 O 2 , as the end product of O 2 − degradation, were suggested to be the main players in the hemin-mediated protein nitration [11,12]. However, the hemin/NO 2 − /H 2 O 2 can be one of the main pathways. ...
Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide (●NO) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the ●NO-binding affinity of hemin has been studied along with the influence of ●NO on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and ●NO, with an investigation of the effects of pH of the reaction medium, generation of H2O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its ●NO-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of ●NO and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and ●NO in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.
... Heme peroxidase-H 2 O 2 -NO 2 − -induced protein tyrosine nitration is a commonly accepted pathway in vivo under inflammatory conditions [4,18,40,41]. So, we firstly examined the peroxidase activity of FeTPPS and used heme as a comparison. As shown in Fig. 2A, FeTPPS exhibited a strong peroxidase activity towards TMB oxidation. ...
... However, FeTPPS is an iron porphyrin compounds that possesses peroxidase activity ( Fig. 2A). Heme peroxidase-H 2 O 2 -NO 2 − system catalyzed protein tyrosine nitration is a commonly accepted pathway in vivo [4,18,40,41,53,54], especially under inflammatory conditions [4,54]. For example, Schildknecht et al. detected a major nitrotyrosine positive protein band around 72 kDa in macrophages by western blotting and identified it as an autocatalytic nitration of prostaglandin endoperoxide synthase-2 (PGHS-2) which was catalyzed by the enzyme's endogenous peroxidase activity in the presence of hydrogen peroxide and nitrite [53,54]. ...
... These metals, and heme, can catalyze the formation of hydroxyl radical, but less so when bound to albumin, and the hydroxyl radical catalyzed from a metal bound to albumin is thought to largely interact with albumin itself rather than damaging other biologically relevant molecules [14,15] (Fig. 1). Iron and heme can also catalyze the nitration of proteins [108][109][110], but when heme is bound to human serum albumin [103,[111][112][113][114] it may facilitate the detoxification of ROS and RNS [115][116][117][118]. The Cys34 on albumin can also form disulfide interactions with glutathione, cysteine, or homocysteine, while Arg410 and Lys525 are main targets of glycation [15,119] (discussed below). ...
... This is also where extravasation of albumin originates thereby resulting in a high concentration at this site in comparison to albumin diffusing away from leaky vessels to other CNS structures and becoming diluted in the process (Fig. 1). Since iron and heme can catalyze reactions leading to oxidation and nitration [108][109][110][123][124][125], it indicates that albumin is positioned to be an early recipient of these reactive species during BBB leakage (Fig. 1). Interestingly, nitrated proteins have been detected around vessels in EAE and MS [40,[126][127][128], and it has been put forth that extravasated albumin from leaky vessels is a main target for nitration during disease [40]. ...
Leakage of the blood–brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth.
... However, it can react to form more dangerous RNS. Upon reaction with ·O 2 -, it gives rise to peroxynitrite (OONO -) and its autoxidation leads to the formation of nitrite (NO 2 -) (Lundberg et al., 2008), both of which can damage biomolecules (Bian et al., 2003;Crawford et al., 2017;Radi, 2018). (1) Molecular oxygen (O 2 ) can be oxidized to superoxide anion (·O 2 -) in the electron transport chain (ETC), by NADPH oxidases (NOX), cytochrome P450 (P450) and other reactions. ...
The emergence and spreading of insecticide resistances in mosquito populations is a major threat to malaria control programs. There is, therefore, an urge to develop new insecticides with novels modes of action. Insects lack the key antioxidant enzyme glutathione reductase, whose role is believed to be compensated by the thioredoxin / thioredoxin reductase system. The aim of my PhD was to study the role of thiroredoxin reductase in the malaria mosquito Anopheles gambiae and to assess its potential as a new insecticidal target. I have established new transgenic redox reporter mosquito lines and shown that, in general, the maintenance of the redo homeostasis is tightly regulated in these species over their entire life cycle. I have further generated CRISPR/Cas9 mutants for thioredoxin reductase and demonstrated that it is essential for mosquito development, but dispensable in the midgut of the adults. Finally, I have identified inhibitors of this enzyme in vitro and shown that they are lethal to Anopheles gambiae mosquitoes.
... As rehearsed above, and in much more detail previously [15,106], while peroxide and superoxide are intermediates, and at high levels are cytotoxic, it is also the hydroxyl radical that causes many or most of the manifestations of oxidative stress and, in particular, of ischaemia-reperfusion injury. Because it is so reactive, it is not observable directly; however, the products of the effective reaction of ROSs with proteins (nitrotyrosine [563][564][565][566][567]), lipids (malondialdehyde [568,569]), and DNA (8-oxoguanine [570][571][572][573][574]) are. Consequently, if we wish to know that I-R injury is a contributor to the kinds of syndromes we are discussing here, we 'simply' need to check whether they manifest these oxidative biomarkers. ...
Ischaemia-reperfusion (I-R) injury, initiated via bursts of reactive oxygen species produced during the reoxygenation phase following hypoxia, is well known in a variety of acute circumstances. We argue here that I-R injury also underpins elements of the pathology of a variety of chronic, inflammatory diseases, including rheumatoid arthritis, ME/CFS and, our chief focus and most proximally, Long COVID. Ischaemia may be initiated via fibrin amyloid microclot blockage of capillaries, for instance as exercise is started; reperfusion is a necessary corollary when it finishes. We rehearse the mechanistic evidence for these occurrences here, in terms of their manifestation as oxidative stress, hyperinflammation, mast cell activation, the production of marker metabolites and related activities. Such microclot-based phenomena can explain both the breathlessness/fatigue and the post-exertional malaise that may be observed in these conditions, as well as many other observables. The recognition of these processes implies, mechanistically, that therapeutic benefit is potentially to be had from antioxidants, from anti-inflammatories, from iron chelators, and via suitable, safe fibrinolytics, and/or anti-clotting agents. We review the considerable existing evidence that is consistent with this, and with the biochemical mechanisms involved.
... The excessive presentation of 3-nitrotyrosine (3-NT) is further increased by the presence of acrylamide in the foods, the alcohol [76], and the iron consumption in the diet [10]. Interestingly, the flavonoids rosmarinic acid [59] and resveratrol [70] reduce the activity of the biomarker glycogen synthase kinase-3 (GSK3B). ...
A large number of plasma proteomic biomarkers have been discovered in the field of neurodegenerative diseases. Novel biomarker molecules in plasma and serum could significantly reduce the need for invasive methods in clinical practice such as the lumbar puncture for CSF collection and may be useful to specific patients. Furthermore, candidate biomarker proteins that have been identified and validated could be used to discriminate Αlzheimer's disease patients from MCI and healthy controls in clinical trials, before the onset of clinical symptoms as well as to improve personalized therapies. The development of new blood-based biomarkers via proteomic technology offers a deep knowledge in the pathophysiology of neurodegenerative diseases and involves in the development of new therapeutic targets. This report presents numerous dietary compounds that either promote or suppress the expression of biomarkers mainly in the blood of AD or MCI subjects.
... The protein nitration pathways induced by ONOO − can be broadly divided into three categories: direct redox reactions, reactions with carbon dioxide (CO 2 ), and homolysis after protonation (Alvarez and Radi, 2003; Figure 1A). Protein nitration via the NO 2 − /H 2 O 2 /heme peroxidase system also involves the formation of oxygen radicals (Bian et al., 2003; Figure 1B). ...
Nitrosative stress, as an important oxygen metabolism disorder, has been shown to be closely associated with cardiovascular diseases, such as myocardial ischemia/reperfusion injury, aortic aneurysm, heart failure, hypertension, and atherosclerosis. Nitrosative stress refers to the joint biochemical reactions of nitric oxide (NO) and superoxide (O 2 – ) when an oxygen metabolism disorder occurs in the body. The peroxynitrite anion (ONOO – ) produced during this process can nitrate several biomolecules, such as proteins, lipids, and DNA, to generate 3-nitrotyrosine (3-NT), which further induces cell death. Among these, protein tyrosine nitration and polyunsaturated fatty acid nitration are the most studied types to date. Accordingly, an in-depth study of the relationship between nitrosative stress and cell death has important practical significance for revealing the pathogenesis and strategies for prevention and treatment of various diseases, particularly cardiovascular diseases. Here, we review the latest research progress on the mechanisms of nitrosative stress-mediated cell death, primarily involving several regulated cell death processes, including apoptosis, autophagy, ferroptosis, pyroptosis, NETosis, and parthanatos, highlighting nitrosative stress as a unique mechanism in cardiovascular diseases.
... Iron is an active metal with a high redox potential that can convert oxidant intermediates such as hydrogen peroxide to harmful oxygen free radicals. In addition, iron has been shown to catalyze the nitration of tyrosine residues, resulting in protein damage [55]. Excess iron can promote oxidative damage-mediated muscle deterioration, leading to muscle atrophy. ...
Sarcopenia is a potential risk factor for weakness, disability and death in elderly individuals. Therefore, seeking effective methods to delay and treat sarcopenia and to improve the quality of life of elderly individuals is a trending topic in geriatrics. Caloric restriction (CR) is currently recognized as an effective means to extend the lifespan and delay the decline in organ function caused by aging. In this review, we describe the effects of CR on improving muscle protein synthesis, delaying muscle atrophy, regulating muscle mitochondrial function, maintaining muscle strength, promoting muscle stem cell (MuSC) regeneration and differentiation, and thus protecting against sarcopenia. We also summarize the possible cellular mechanisms by which CR delays sarcopenia. CR can delay sarcopenia by reducing the generation of oxygen free radicals, reducing oxidative stress damage, enhancing mitochondrial function, improving protein homeostasis, reducing iron overload, increasing autophagy and apoptosis, and reducing inflammation. However, the relationships between CR and genetics, sex, animal strain, regimen duration and energy intake level are complex. Therefore, further study of the proper timing and application method of CR to prevent sarcopenia is highly important for the aging population.
... The main peroxynitrite-independent mechanism involved in tyrosine oxidation and nitration requires three participants: H 2 O 2 , NO 2 − and a heme peroxidase (or free heme) [71][72][73]. For this system, tyrosine nitration depends on the initial reaction between the heme peroxidase (HP) and H 2 O 2 , in a two electron reaction that reduces H 2 O 2 to H 2 O ...
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.
... Nitration occurs via simultaneous oxidation of nitrite to nitrogen dioxide radical and tyrosine to tyrosyl radical, respectively (Bian et al. 2003, Brennan et al. 2002, Eiserich et al. 1998, Pfeiffer et al. 2001, van der Vliet et al. 1997. The described reaction plays a major role in host defense mechanisms, as activated phagocytes contain high levels of the abovementioned peroxidases, especially myeloperoxidases and eosinophil peroxidases (Gaut et al. 2002, Wu et al. 1999. ...
... [2] The peroxynitrite-dependent and-independentp athways for protein tyrosine nitration are induced under biological conditions by superoxide dismutase (Cu-Zn-SOD [3] andM n-SOD) [4] and heme-containing proteins, respectively. [5] In addi-tion, in the presence of H 2 O 2 ,m etal ions (Fe II and Cu II )g enerate hydroxyl radicals (HOC)t hat oxidizeN O 2 À to CNO 2 by Fenton reactions,l eadingt ot he production of 3-NT. [6,7] So far,m ost of the studies have been focusedo nF e-, Cu-, and Mn-based tyrosine nitration. ...
The nitration of tyrosine residues in proteins represents a specific footprint of the formation of reactive nitrogen species (RNS) in vivo. Here, the fusion product of orange protein (ATCUN‐ORP) was used as an in vitro model system containing an amino terminal Cu(II)‐ and Ni(II)‐binding motif (ATCUN) tag at the N‐terminus and a native tyrosine residue in the metal‐cofactor‐binding region for the formation of 3‐NO2‐Tyr (3‐NT). It is shown that NiII‐ATCUN unusually performs nitration of tyrosine at physiological pH in the presence of the NO2⁻/SO3²⁻/O2 system, which is revealed by a characteristic absorbance band at 430 nm in basic medium and 350 nm in acidic medium (fingerprint of 3‐NT). Kinetics studies showed that the formation of 3‐NT depends on sulfite concentration over nitrite concentration suggesting key intermediate products, identified as oxysulfur radicals, which are detected by spin‐trap EPR study by using 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO). This study describes a new route in the formation of 3‐NT, which is proposed to be linked with the sulfur metabolism pathway associated with the progression of disease occurrence in vivo.
... Heme is degraded by the rate-limiting enzyme HO-1 that possesses antioxidant function and promotes many biological oxidation processes involved in oxygen transport, mitochondrial respiration, cellular antioxidant defenses, and signal transduction processes. For these reasons heme proteins are considered a source of ROS and their acute exposure aggravates oxidative injury [101,102]. The formation of H 2 O 2 in erythrocytes is associated with the autoxidation of oxyhemoglobin, a process that produces superoxide, which, in turn, reacts with SOD enzymes producing H 2 O 2 and oxygen. ...
Hydrogen peroxide (H2O2) is an important metabolite involved in most of the redox metabolism reactions and processes of the cells. H2O2 is recognized as one of the main molecules in the sensing, modulation and signaling of redox metabolism, and it is acting as a second messenger together with hydrogen sulfide (H2S) and nitric oxide (NO). These second messengers activate in turn a cascade of downstream proteins via specific oxidations leading to a metabolic response of the cell. This metabolic response can determine proliferation, survival or death of the cell depending on which downstream pathways (homeostatic, pathological, or protective) have been activated. The cells have several sources of H2O2 and cellular systems strictly control its concentration in different subcellular compartments. This review summarizes research on the role played by H2O2 in signaling pathways of eukaryotic cells and how this signaling leads to homeostatic or pathological responses.
... Numerous studies have established that cancer tissue cells have Fenton reactions (Stevens and Kalkwarf, 1990;Toyokuni, 2009;Akatsuka et al., 2012). Iron-sulfur clusters (Johnson et al., 2005), heme (Bian et al., 2003), and labile iron pool (LIP) (Kruszewski, 2003) have been identified as sources of Fe 2+ for cellular Fenton reactions. A variety of molecules can reduce Fe 3+ to Fe 2+ , such as NAD(P)H, superoxide ðO • − 2 Þ, S 2− , and ascorbic acid, which can lead to repeated Fenton reactions, also called the Haber-Weiss reaction (Fong et al., 1976;Kojima and Bates, 1979;Elmagirbi et al., 2012), giving rise to continuous production of •OH and OH − if the cellular environment is rich in such molecules. ...
We present a computational study of tissue transcriptomic data of 14 cancer types to address: what may drive cancer cell division? Our analyses point to that persistent disruption of the intracellular pH by Fenton reactions may be at the root of cancer development. Specifically, we have statistically demonstrated that Fenton reactions take place in cancer cytosol and mitochondria across all the 14 cancer types, based on cancer tissue gene-expression data integrated via the Michaelis-Menten equation. In addition, we have shown that (i) Fenton reactions in cytosol of the disease cells will continuously increase their pH, to which the cells respond by generating net protons to keep the pH stable through a combination of synthesizing glycolytic ATPs and consuming them by nucleotide syntheses, which may drive cell division to rid of the continuously synthesized nucleotides; and (ii) Fenton reactions in mitochondria give rise to novel ways for ATP synthesis with electrons ultimately coming from H2O2, largely originated from immune cells. A model is developed to link these to cancer development, where some mutations may be selected to facilitate cell division at rates dictated by Fenton reactions.
... This nitryl chloride is a potent nitrating agent. Certain metalloproteins comprising hemoglobin, cytochrome c, superoxide dismutase catalyze H 2 O 2 dependent oxidation of nitrite to form 3-nitrotyrosine [44]. ...
... Nitric oxide itself can be harmful to the cell because it has the ability to form powerful reactive nitrogen species (ONOO -, NO 2 , NO 2 O 3 ) with the end products of cellular respiration (superoxides, hydroxyl radicals, and hydrogen peroxides), called ROS. Furthermore, reactive nitrogen species have been documented to take part in neuronal apoptosis (Bian et al. 2003). ...
... The nitration of Tyr is mediated by RNS such as ONOO -/ONOOH and • NO 2 although nitration can also by accomplished by heme peroxidases and nitrite [20]. The two main mechanisms of biological nitration, the ONOO -/ONOOH and the heme peroxidase pathways, lead both to the formation of Tyr • and • NO 2 , which combine with diffusion controlled rates to form 3nitrotyrosine (3-NT; Fig. 1H). ...
Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.
... Rabbit protein lacks this N-linked oligosaccharide. In vitro studies support that heme can catalyze protein nitration in the presence of H2O2 and nitrite (NO 2-), the stable end products of NO and O2degradation (36,37). Based on these published studies with heme bound to bovine serum albumin, heme in vivo (such as that bound to the weak, not the tight, binding site on human serum albumin) may well help catalyze nitration as part of heme toxicity in hemolytic conditions. ...
Hemopexin protects against heme toxicity in hemolytic diseases and conditions, sepsis, and sickle cell disease. This protection is sustained by heme-hemopexin complexes in biological fluids that resist oxidative damage during heme-driven inflammation. However, apo-hemopexin is vulnerable to inactivation by reactive nitrogen (RNS) and oxygen species (ROS) that covalently modify amino acids. The resultant nitration of amino acids is considered a specific effect reflecting biological events. Using LC-MS, we discovered low endogenous levels of tyrosine nitration in the peptide YYCFQGNQFLR in the heme-binding site of human hemopexin, which was similarly nitrated in rabbit and rat hemopexins. Immunoblotting and selective reaction monitoring were used to quantify tyrosine nitration of in vivo samples and when hemopexin was incubated in vitro with nitrating nitrite/myeloperoxidase/glucose oxidase. Significantly, heme binding by hemopexin declined as tyrosine nitration proceeded in vitro. Three nitrated tyrosines reside in the heme-binding site of hemopexin, and we found that one, Tyr-199, interacts directly with the heme ring D propionate. Investigating the oxidative modifications of amino acids after incubation with tert-butyl hydroperoxide and hypochlorous acid in vitro, we identified additional covalent oxidative modifications on four tyrosine residues and one tryptophan residue of hemopexin. Importantly, three of the four modified tyrosines, some of which have more than one modification, cluster in the heme-binding site, supporting a hierarchy of vulnerable amino acids. We propose that during inflammation, apo-hemopexin is nitrated and oxidated in niches of the body containing activated RNS- and ROS-generating immune and endothelial cells, potentially impairing hemopexin’s protective extracellular antioxidant function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
... The wide-known function of iron is the contribution in heme molecule synthesis, copper is mandatory to iron uptake in hemoglobin synthesis operation (Deger et al., 2005). Iron deficiency may lead to hypochromic microcytic anemia (Bainton and Finch 1964; Bian et al., 2003). In recent years, several researches pinpoint the role of highly reactive oxygen radicals in pathogenesis of equine babesiosis (Deger et al., 2009), but still scarce papers discussing this topic. ...
Equine piroplasmosis is a clinically significant widespread tick borne disease affecting equine population. Thus, this study aims to describe the clinical signs, correlation between the developed anemias and mineral status along with the lipid peroxidation product Malondialdehyde (MDA) in naturally occurring T. equi infected horses in Egypt. Twentyfive
horses of different age and sex were involved in this study; horses have signs compatible with babesial infection were examined. Fever, hemoglobinuria and icterus were the most consistent clinical signs recorded in this study. The hematology picture showed decrease in red cell parameters along with leucocytosis. Macrocytic hypochromic anemia was found in infected horses alongside relative increase in copper and relative decrease in ferrous and zinc. MDA showed very statistically significant difference when compared to control data. In conclusion, high level of MDA along with reduction in PCV, HB, and RBCs count is indicating the presence of oxidative stress and implicating the process as a cause of anemia in T. equi infection. The mineral status (Cu, Zn, Fe) appeared to be directly affected by the parasite and the mineral status influenced by the anemic syndrome associated with T. equi. Inversely correlation between zinc and MDA level might be used in planning the treatment strategy of T. equi. Including these minerals in treatment regimen of T. equi will help to counter the anemic nature of the disease.
... To understand the effect of diaryl selenides 15-21 and spirodiazaselenuranes 29-35 against PN, the PN-scavenging activity in PN-mediated nitration of tyrosyl residues in bovine serum albumin (BSA) was studied [40,41]. The inhibition of nitration of tyrosine residues was followed by immunoblotting methods using antibody against 3-nitro-L-tryrosine. ...
Spirodiazaselenuranes are structurally interesting compounds and the stability of these compounds depends highly on the nature of the substituents attached to the nitrogen atoms. Aromatic substituents are known to play important roles in stabilizing the Se-N bonds in spiro compounds. In this study, several spirodiazaselenuranes are synthesized by introducing benzylic and aliphatic substituents to understand their effect on the stability of the Se-N bonds and the antioxidant activity. Replacement of phenyl substituent by benzyl/alkyl groups significantly reduces the stability of the spirodiazaselenuranes and slows down the oxidative cyclization process. The selenium centre in the spiro compounds undergoes further oxidation to produce the corresponding selenurane oxides, which are stable at room temperature. Comparison of the glutathione peroxidase (GPx) mimetic activity of the compounds showed that the diaryl selenides having heterocyclic rings are significantly more active due to the facile oxidation of the selenium centre. However, the activity is reduced significantly for compounds having aliphatic substituents. In addition to GPx activity, the compounds also inhibit peroxynitrite-mediated nitration and oxidation reaction of protein and small molecules, respectively. The experimental observations suggest that the antioxidant activity is increased considerably upon substitution of the aromatic group with the benzylic/aliphatic substituents on the nitrogen atoms.
... Some reports suggested that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility. Unlike that, severe oxidative stress diminishes intracellular proteolysis by generating severely damaged, crosslinked proteins that cannot be easily degraded and also by damaging proteasome proteolytic enzymes [2,10]. MnSOD is particularly sensitive to oxidative inactivation and posttranslational modifications [8]. ...
Previously, we examined manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), and catalase (CAT) activities in rat brain irradiated with 2 or 3 Gy of γ-rays. The results indicated that lower MnSOD activity and inducibility found in hippocampus might explain higher radiosensitivity of this brain region. Thus, in this study, we wanted to determine changes of MnSOD, CuZnSOD, and CAT activities after dose of 5 Gy and to find out if differences in MnSOD activity are caused by changes in its expression.
Heads of 4-day-old female rats were irradiated with γ-rays, using (60)Co. Animals were sacrificed 1/24 h after exposure. Hippocampus and cortex tissues were prepared for enzyme activity measurements and Western blot analysis.
One hour after exposure, γ-rays significantly decreased MnSOD activity in both examined brain regions. Twenty-four hours later, MnSOD recovery showed dose and regional dependence. It was weaker at higher doses and in hippocampal region. MnSOD expression changed in the similar manner as MnSOD activity only at lower doses of γ-rays. In both examined brain regions, gamma radiation significantly decreased CuZnSOD activity and did not change activity of CAT.
Our results confirmed that MnSOD plays an important role in different regional radiosensitivity but also showed that depending on dose, radiation affects MnSOD level by utterly different mechanisms. Postradiation changes of CuZnSOD and CAT are not regionally specific and therefore, cannot account for the different radiosensitivity of the hippocampus and cortex.
Vasoactive intestinal peptide (VIP) is a neuropeptide that play an important role in immunoregulation and anti-inflammation. Numerous inflammatory/autoimmune disorders are associated with decreased VIP binding ability to receptors and diminished VIP activation of cAMP generation in immune cells. However, the mechanisms linking oxidative/nitrative stress to VIP immune dysfunction remain unknown. It has been reported that the elevated heme or Cu2+ in inflammatory diseases can cause oxidative and nitrative damage to nearby biological targets under high oxidative stress conditions, which affects the structure and activity of linked peptides or proteins. Thus, the VIP down-regulated immune response may be interfered by redox metal catalyzed VIP tyrosine nitration. To explore this, we systematically investigated the possibility of heme or Cu2+ to catalyze VIP tyrosine nitration. The results showed that Tyr10 and Tyr22 of VIP can both be nitrated in heme/H2O2/NO2- system as well as in Cu2+/H2O2/NO2- system. Then, we used synthetic mutant VIPs with tyrosine residues substituted by 3-nitrotyrosine to study the impact of tyrosine nitration on VIP activity in SHSY-5Y cells. Our findings demonstrated that VIP nitration dramatically decreased the content of its α-helix and random coil, suggesting that VIP nitration might reduce its affinity to the receptor. This was further confirmed in the cAMP assay. The results showed that 10 nM of these tyrosine nitrated VIPs could significantly (p < 0.01) decrease cAMP secretion compared to the wild type VIP. Our data reveal that the attenuation of the neuroprotective effect of VIP in inflammation-related diseases might be attributed to metal-catalyzed VIP tyrosine nitration.
The development of a manageable reactive nitrogen species-potentiated nitrosative stress induction system for cancer therapy has remained elusive. Herein, tailored silica-based nanoscintillators were reported for low-dosage X-ray boosting for the in situ formation of highly cytotoxic peroxynitrite (ONOO-). Significantly, cellular nitrosative stress revolving around the intracellular protein tyrosine nitration through ONOO- pathways was explored. High-energy X-rays were directly deposited on silica-based nanoscintillators, forming the concept of an open source and a reduced expenditure-aggravated DNA damage strategy. Moreover, the resultant ONOO-, along with the released nitric oxide, not only can act as "oxygen suppliers" to combat tumor hypoxia but also can induce mitochondrial damage to initiate caspase-mediated apoptosis, further improving the therapeutic efficacy of radiotherapy. Thus, the design of advanced nanoscintillators with specific enhanced nitrosative stress offers promising potential for postoperative radiotherapy of colon cancer.
This study investigated a novel sodium iron chlorophyllin-H2O2 (SIC–H2O2) sludge pretreatment strategy before anaerobic digestion to enhance methane production. The efficiencies and mechanism of the proposed strategy to enhance sludge biodegradability were explored. The SIC–H2O2 pretreatment could enhance the oxidation performance for sludge floc disintegration to dissociate TB-EPS into S-EPS increased SCOD to 521.38 mg/L. The increase of solubilization and release of EPS with the pretreatment facilitate the biogas production at 702 L kg⁻¹ VS, which was 3-folds of the control and significantly higher than other pretreatments. The result of excitation–emission matrix and parallel factor (EEM-PARAFAC) analysis showed that the SIC–H2O2 pretreatment enhanced the dissociation of TB-EPS fractions, especially the protein-like and soluble microbial by-product-like substances. Electron paramagnetic resonance (EPR) results provided evidence for homolytic catalysis H2O2 for the generation OH and the production of high-valent (Por)FeIV(O) intermediates. Synergistic effects of reactive oxygen species (OH, H2O2 and O2⁻/HO2) and (Por)FeIV(O) enhanced the EPS disintegration during SIC–H2O2 pretreatment. The mixed-acid type fermentation provided continuous VFAs supply under the enrichment of Chloroflexi and Actinobacteria and multiplication Methanosaeta also promoted methane production. This research provides a feasible pretreatment strategy increase sludge biodegradability and enhance biogas production in the anaerobic digestion process.
X-ray-triggered nitrite (NO2⁻) is used for hypoxic prostate cancer therapy by inhibiting protective autophagy and inducing lethal nitrosative stress based on ZIF-82-PVP. After entering tumor cells, the released 2-nitroimidazole ligands capture low-energy electrons derived from X-rays to produce NO2⁻, and the Zn²⁺ inhibits the migration and invasion of tumor cells.
Abstract
Although reactive oxygen species (ROS)-mediated tumor treatments are predominant in clinical applications, ROS-induced protective autophagy promotes cell survival, especially in hypoxic tumors. Herein, X-ray triggered nitrite (NO2⁻) is used for hypoxic prostate cancer therapy by inhibiting autophagy and inducing nitrosative stress based on an electrophilic zeolitic imidazole framework (ZIF-82-PVP). After internalization of pH-responsive ZIF-82-PVP nanoparticles, electrophilic ligands and Zn²⁺ are delivered into cancer cells. Electrophilic ligands can not only consume GSH under hypoxia but also capture low-energy electrons derived from X-rays to generate NO2⁻, which inhibits autophagy and further elevates lethal nitrosative stress levels. In addition, dissociated Zn²⁺ specifically limits the migration and invasion of prostate cancer cells through ion interference. In vitro and in vivo results indicate that ZIF-82-PVP nanoparticles under X-ray irradiation can effectively promote the apoptosis of hypoxic prostate cancer cells. Overall, this nitrosative stress-mediated tumor therapy strategy provides a novel approach targeting hypoxic tumors.
Although reactive oxygen species (ROS)‐mediated tumor treatments are predominant in clinical applications, ROS‐induced protective autophagy promotes cell survival, especially in hypoxic tumors. Herein, X‐ray triggered nitrite (NO2⁻) is used for hypoxic prostate cancer therapy by inhibiting autophagy and inducing nitrosative stress based on an electrophilic zeolitic imidazole framework (ZIF‐82‐PVP). After internalization of pH‐responsive ZIF‐82‐PVP nanoparticles, electrophilic ligands and Zn²⁺ are delivered into cancer cells. Electrophilic ligands can not only consume GSH under hypoxia but also capture low‐energy electrons derived from X‐rays to generate NO2⁻, which inhibits autophagy and further elevates lethal nitrosative stress levels. In addition, dissociated Zn²⁺ specifically limits the migration and invasion of prostate cancer cells through ion interference. In vitro and in vivo results indicate that ZIF‐82‐PVP nanoparticles under X‐ray irradiation can effectively promote the apoptosis of hypoxic prostate cancer cells. Overall, this nitrosative stress‐mediated tumor therapy strategy provides a novel approach targeting hypoxic tumors.
Understanding the toxicological properties of MnIII-porphyrins (MnTPPS, MnTMPyP or MnTBAP) can provide important biochemical rationales in developing them as the therapeutic drugs against protein tyrosine nitration induced inflammation diseases. Here, we present a comprehensive understanding of the pH-dependent redox behaviors of these MnIII-porphyrins and their structural effects on catalyzing bovine serum albumin (BSA) nitration in the presence of H2O2 and NO2−. It was found that both MnTPPS and MnTBAP stand out in catalyzing BSA nitration at physiologically close condition (pH 8), yet they are less effective at pH 6 and 10. MnTMPyP was shown no ability to catalyze BSA nitration under all tested pHs (pH 6, 8 and 10). The kinetics and active intermediate determination through electrochemistry method revealed that both the pH-dependent redox behavior of the central metal cation and the antioxidant capability of porphin derivative contribute to the catalytic activities of three MnIII-porphyrins in BSA nitration in the presence of H2O2/NO2−. These comprehensive studies on the oxidative reactivity of MnIII-porphyrins towards BSA nitration may provide new clues for searching the manganese based therapeutic drugs against the inflammation related diseases.
Many metal-organic frameworks have been designed and synthesized for biosensor due to high surface area and porosity, suitable size and good biocompatibility. Despite recent advances, however, most of them are only as the nanocarrier. In this work, a new artificial nanozyme was constructed on the metalloporphyrinic metal organic frameworks (PMOF(Fe)), which was formed by Fe porphyrin and Zr4+ ions. Then ultra-small Pt nanoparticles (Pt NPs) was loaded on the surface of PMOF(Fe) to form [email protected](Fe). Because of PMOF(Fe) with high surface area and exposed Fe activity centre, PMOF(Fe) works as the nanocarrier to hinder the Pt NPs aggregating and exhibits high peroxidase mimics activity. So, Pt NPs decorated on the surface of PMOF(Fe) possessed high stability and exhibited high activity. Due to the synergistic effect between PMOF(Fe) and Pt NPs, [email protected](Fe) exhibits superior catalase-like and peroxidase-like activity. Moreover, [email protected](Fe) possesses high electrocatalytic activity towards to the reduction of H2O2 and oxygen reduction reaction (ORR). This strategy may serve as a strong foundation to design MOF-based artificial nanozyme and develop an ideal platform for MOF and nanozymes toward artificial enzymatic catalytic systems, fuel cell and new analytical applications.
Water-soluble iron porphyrins, such as FeTPPS (5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III)), FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphyrinato iron (III) chloride) and FeTBAP (5,10,15,20-tetrakis (4-benzoic acid) porphyrinato iron (III)), are highly active catalysts for peroxynitrite decomposition and thereby have been suggested as therapeutic agent for inflammatory diseases that implicate the involvement of nitrotyrosine formation. Here, we systemically investigated catalytic properties of FeTPPS, FeTMPyP and FeTBAP on protein nitration in the presence of hydrogen peroxide and nitrite. We showed that FeTPPS, FeTBAP and FeTMPyP all exhibited higher peroxidase activity in compared with hemin. As to protein nitration, the catalytic effect of FeTPPS and FeTBAP are effective in the presence of hydrogen peroxide and nitrite, while negligible BSA nitration was observed in the case of FeTMPyP. Moreover, the underlying mechanism of the oxidation of FeTPPS, FeTBAP and FeTMPyP was further studied. Collectively, our results suggest that, compound I and II species are involved in as the key intermediates in FeTMPyP/H2O2 system as similar as those in FeTPPS/H2O2 and FeTBAP/H2O2 system. As compared to weak antioxidants, TPPS and TBAP, however, TMPyP scavenges oxo-Fe (IV) intermediates of FeTMPyP at a faster rate by significant self-degradation; results in the shortest lifetimes of OFeIV-TMPyP and the lowest catalytic activity on oxidizing tyrosine and nitrite; and therefore, attributes to inactivation of FeTMPyP in protein nitration. In addition, association of FeTMPyP to BSA was found weak, while strong binding of FeTPPS and FeTBAP were observed. The weak binding keeps away of target residue of BSA from the center of FeTMPyP where the RNS is generated, which might be attributed as additional factors to the inactivation of FeTMPyP in protein nitration.
The Fenton reaction was discovered over 120 years ago, yet our understanding of the complete reaction mechanism of the seemingly simple iron and hydrogen peroxide reaction (Fe+H2O2) remains unclear, thus limiting its full potential. In this work, the aim is to summarize the processes that pertain to the (photo)Fenton reaction. More specifically, this review does not consider previous work relating to the Advanced Oxidation Process (AOP)-mediated disinfection/decontamination of stream flows, as much work has successfully achieved this. Instead, this manuscript presents an overview of the other fields of environmental and medical applications of (photo)Fenton: i) surface pre-treatment (nano-particles functionalization or modification), ii) terrestrial and algal biomass (pre)treatment to enhance high value products’ recovery, iii) cancer treatment (malignant tumor elimination) and iv) other medical uses (antibiotics development, wounds disinfection, root canal sterilization and teeth whitening). For each field of application, the focus lies on analyzing the conceptualization and framework of the (photo)Fenton process, the opportunities and limitations, as well as a series of related applications. To conclude, the work discusses the current limitations of the existing applications and future research avenues.
Peroxynitrite-mediated nitrosative stress in the brain has been associated with various neurodegenerative disorders. Recent evidence highlights peroxisome proliferator-activated receptor γ (PPAR γ ) as a critical neuroprotective factor in neurodegenerative diseases. Here, we observed the effect of the herb hydroxysafflor yellow A (HSYA) during nitrosative stress in neurons and investigated the mechanism based on PPAR γ protection. We found that a single exposure of primary neurons to peroxynitrite donor SIN-1 caused neuronal injury, which was accompanied by the increase of PPAR γ nitration status and lack of activation of the receptor, as measured by PPAR γ DNA-binding activity, by agonist (15d-PGJ2 or rosiglitazone) stimulation. The crucial role of PPAR γ in neuronal defense against nitrosative stress was verified by showing that pretreatment with 15d-PGJ2 or rosiglitazone attenuated SIN-1-induced neuronal injury but pretreatment with GW9662, a PPAR γ antagonist, aggravated SIN-1-induced neuronal injury. The addition of HSYA not only inhibited SIN-1-induced neuronal damage but prevented PPAR γ nitrative modification and resumed PPAR γ activity stimulated by either 15d-PGJ2 or rosiglitazone. Furthermore, HSYA also showed the ability to rescue the neuroprotective effect of 15d-PGJ2 or rosiglitazone when the agonists were coincubated with SIN-1. Finally, in vivo experiments demonstrated that the administration of HSYA also efficiently blocked PPAR γ nitration and loss of activity in the SIN-1-injected hippocampus and reversed the increased neuronal susceptibility which was supported by the inhibition of Bcl-2 protein downregulation induced by SIN-1. The results suggest that HSYA protects neurons from nitrosative stress through keeping PPAR γ as a functional receptor, allowing a more effective activation of this neuroprotective factor by the endogenous or exogenous agonist. Our findings provide new clues in understanding the role of the neuroprotective potential of the herbal HSYA.
As widely distributed domestic animals, sheep are an important species and the source of mutton. In this study, we aimed to evaluate the regulatory lncRNAs associated with muscle growth and development between high production mutton sheep (Dorper sheep and Qianhua Mutton Merino sheep) and low production mutton sheep (Small-tailed Han sheep). In total, 39 lncRNAs were found to be differentially expressed. Using co-expression analysis and functional annotation, 1,206 co-expression interactions were found between 32 lncRNAs and 369 genes, and 29 of these lncRNAs were found to be associated with muscle development, metabolism, cell proliferation and apoptosis. lncRNA–mRNA interactions revealed 6 lncRNAs as hub lncRNAs. Moreover, three lncRNAs and their associated co-expressed genes were demonstrated by cis-regulatory gene analyses, and we also found a potential regulatory relationship between the pseudogene lncRNA LOC101121401 and its parent gene FTH1. This study provides a genome-wide resolution of lncRNA and mRNA regulation in muscles from mutton sheep.
Heme proteins can both form and metabolize nitric oxide (NO). The predominant mechanism for NO scavenging by heme proteins is the dioxygenation reaction where NO reacts with oxygenated ferrous heme proteins, particularly hemoglobin and myoglobin, to form ferric heme and nitrate. The degree to which NO is scavenged by hemoglobin is largely limited by encapsulation of the hemoglobin in the red blood cell. With red blood cell hemolysis, the release of hemoglobin into plasma will reduce NO concentrations and impair endothelial-dependent NO signaling. NO binding to ferrous heme proteins also affects NO signaling and inhibits mitochondrial respiration by direct inhibition of cytochrome c oxidase. In contrast, nitrite can react with deoxygenated ferrous heme proteins to form NO. Particular attention is paid to the mechanisms of nitrite reduction by hemoglobin and to the evidence that intraerythrocytic hemoglobin bioactivates nitrite, driving NO signaling under physiological and pathological hypoxia. Heme proteins thus regulate NO bioavailabilty via oxidative and reductive reactions with NO and nitrite, respectively.
Neuropeptide Y (NPY) is a member of the pancreatic peptide family of neuropeptides that play a crucial role in numerous central and peripheral nervous system responses. Recently, it has been shown that NPY protected cells against neurotoxic damage from β-amyloid peptides (Aβ) in Alzheimer's disease (AD). Heme is a common factor linking several metabolic perturbations in AD and altered heme metabolism has been shown a relationship to the pathologies of AD. Thus, heme may have a chance to meet NPY and potentially counteract its function. To explore this, UV-Visible spectroscopy, fluorescence spectroscopy and differential pulse voltammetry (DPV) were used to demonstrate that NPY can bind with heme to form a NPY-heme complex and the binding enhances the peroxidase activity of heme. Dot blotting result indicates that NPY is easily to be nitrated by binding with heme when H2O2 and NO2- are present. Furthermore, LC-MS/MS results confirm that tyrosine36 (Y36), an important amino acid residue of NPY in binding and activating for neuropeptide receptors, can be nitrated during the nitration process. Thereafter, we used mutant peptide NPY（3N）with Y36 replaced by 3-nitrtotyrosine to investigate the impact of nitration on the structure and bioactive of the peptide. Our results show that Y36 nitration destabilizes the α-helix conformation of the peptide, and counteract NPY-induced inhibition of cAMP accumulation in SK-N-MC cells. Collectively, these data imply that the self-association of NPY with heme potentially induces tyrosine nitration, destroys active monomeric conformation of the peptide and thereby counteracts its bioactivity
Reaction of a Ni(II) complex of ligand L (L = meso-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane) (1) in methanol with NO2 has been studied. Addition of equivalent amount of NO2 resulted in the reduction of Ni(II) to Ni(I). The reduction was monitored by UV-visible, EPR spectroscopy. The Ni(I) intermediate reacted with further equivalent of NO2 to form Ni(II)-O-nitrito complex, [NiII(L)(η¹-ONO)]⁺ (2). Structural characterization confirmed the formation of 2. Subsequent equivalent of NO2 resulted in the oxo transfer from NO2 to the metal bound nitrito group leading to the formation of O-nitrato complex, [NiII(L)(η¹-ONO2)]⁺ (3) with simultaneous release of NO. The release of NO in the reaction was evidenced by spin trapping experiment. Complex 3 was isolated and characterized structurally.
The human peroxidases are hemoproteins that utilize H2O2 to oxidize a variety of endogenous and exogenous substrates. The established members of this enzyme family are eosinophil peroxidase, lactoperoxidase, myeloperoxidase, and thyroid peroxidase. The reactions catalyzed by these enzymes, which include halide ion oxidation and substrate free radical formation, contribute to cellular pathology and xenobiotic toxicity. In contrast to the peroxidases, the peroxiredoxins are cysteine-containing proteins that detoxify H2O2 and alkyl peroxides at the expense of electron donors such as thioredoxin and cellular thiols. They do not generally oxidize other endogenous or xenobiotic substrates. The peroxiredoxins appear to be constituents of oxidative stress signaling pathways and help to ameliorate oxidative stress, but do not contribute significantly to xenobiotic toxicity.
Ferritin is a giant protein composed of 24 subunits which is able to sequester up to 4500 atoms of iron. We proposed two kinds of heme binding sites in mammalian ferritins and provided direct evidence for peroxidase activity of heme-ferritin, since there is the possibility that “ferritin–heme” systems display unexpected catalytic behavior like heme-containing enzymes. In the current study, peroxidase activity of heme-bound ferritin was studied using TMB1, L-DOPA, serotonin, and dopamine, in the presence of H2O2, as oxidant substrate. The catalytic oxidation of TMB was consistent with first-order kinetics with respect to ferritin concentration. Perturbation of the binding affinity and catalytic behavior of heme-bound His-modified ferritin were also documented. We also discuss the importance of the peroxidase-/nitrative-mediated oxidation of vital molecules as well as ferritin-induced catalase inhibition using in vitro experimental system. Uncontrollable “heme–ferritin”-based enzyme activity as well as up-regulation of heme and ferritin may inspire that some oxidative stress-mediated cytotoxic effects in AD-affected cells could be correlated to ferritin-heme interaction and/or ferritin-induced catalase inhibition and describe its contribution as an important causative pathogenesis mechanism in some neurodegenerative disorders.
Subtilisin QK, which is newly identified as a fibrinolytic enzyme from Bacillus subtilis QK02, has the ability of preventing nitrotyrosine formation in bovine serum albumin induced by nitrite, hydrogen peroxide and hemoglobin in vitro verified by ELISA, Western-blot and spectrophotometer assay. Subtilisin QK also attenuates the fluorescence emission spectra of bovine serum albumin in the course of oxidation caused by nitrite, hydrogen peroxide and hemoglobin. Furthermore, subtilisin QK could suppress the transformation of oxy-hemoglobin to met-hemoglobin caused by sodium nitrite, but not the heat-treated subtilisn QK. Compared with some other fibrinolytic enzymes and inactivated subtilisin QK treated by phenylmethylsulfonylfluoride, the ability of inhibiting met-hemoglobin formation of subtilisin QK reveals that the anti-oxidative ability of subtilisin QK is not concerned with its fibrinolytic function. Additionally, nitrotyrosine formation in proteins from brain, heart, liver, kidney, and muscle of mice that is intramuscular injected the mixture of nitrite, hydrogen peroxide and hemoglobin is attenuated by subtilisin QK. Subtilisin QK can also protect Human umbilical vein endothelial cell (ECV-304) from the damage caused by nitrite and hydrogen peroxide.
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.
Protein tyrosine nitration is a biomarker of NO-dependent oxidative stress. Protein nitration will induce the changes of protein catalytic activities, cell signaling and cell skeletal structure, and lead to relevant pathological events. In this paper, the roles of iron in different pathways of protein nitration are introduced, and the results indicated that microelement iron in vivo plays an important role in protein nitration.
Nitrite anions are key small molecules for regulating many physiological processes, and are abundant in blood and tissues. In the present work, the reactivity of α-NH 2 of N-terminus and ε-NH 2 of lysine of peptides and proteins toward nitrous acids was studied using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The synthesized standard peptides and standard protein (myoglobin) were used as model system to investigate the effect of different reaction system and different time on the reaction degree of peptides with nitrous acid. The results show that the diazo-reaction occurs between amino group of N-teminal of peptides and nitrous acid, and the intermediate formed in this reaction is a diazonium ion that is unstable to give nitrogen gas and a carbocation. The carbocation proceeds either to nucleophilic substitution or to elimination. The product of M +1 is formed by substitution. The other major product of M-17 observed in the present study is the alkene derivative formed by an elimination reaction. When the reaction system concludes acetic acid and sodium nitrite, the nitrous acid can completely react with amino group of N-termial peptides in 5 min. However,in the same situation, ε-NH 2 of lysine of peptides could not react with nitrous acids. The results of this study can provide useful information for understanding the interaction of nitrous acid and proteins in depth.
The human peroxidases are hemoproteins that utilize H2O2 to oxidize a variety of endogenous and exogenous substrates. The well-established members of this enzyme family are eosinophil peroxidase, lactoperoxidase, myeloperoxidase, and thyroid peroxidase. The reactions catalyzed by these enzymes, which include halide oxidation and substrate free radical formation, contribute to cellular pathology and xenobiotic toxicity. In contrast to the peroxidases, the peroxiredoxins are cysteine-containing proteins that detoxify both H2O2 and alkyl peroxides at the expense of electron donors such as thioredoxin and cellular thiols. They do not oxidize other endogenous or xenobiotic substrates. The peroxiredoxins appear to be constituents of oxidative stress signaling pathways and help to ameliorate oxidative stress, but do not appear to contribute significantly to xenobiotic toxicity.
Cu(ii) complexes of N2O2 type ligands, L(1)H2 and L(2)H2 [L(1)H2 = 6,6'-(((pyridin-2-ylmethyl)azanediyl)bis(methylene))bis(2,4-di-tert-butylphenol); L(2)H2 = 2,4-di-tert-butyl-6-(((3-(tert-butyl)-2-hydroxy-5-methylbenzyl)(pyridin-2-yl-methyl)amino)methyl)phenol], have been synthesized. Addition of nitrogen dioxide (NO2) in THF solutions of the complexes resulted in the nitration at the 4-position of a coordinated equatorial phenolate ring of the ligand frameworks. This nitration did not occur at the phenol ring which is axially coordinated to the metal center. Spectroscopic evidence suggests that the reaction proceeds through a phenoxyl radical complex formation.
The heading title of the article [Phosphorus Research Bulletin 26, 39-52 (2012)] was incorrect because of the editor's mistake on the volume number. The corrected volume number of the heading title is "Vol.26".
A novel label-free method for the in-situ monitoring of protein tyrosine nitration (PTN) was explored based on surface-enhanced Raman spectroscopy (SERS). Benefiting from the relative weak binding ability of sulfate to silver surface, the Raman signals of nitrated peptides were boosted well with sulfate-aggregated silver nanoparticles (Ag NPs). The distinction of the SERS spectra between non-nitrated peptides and nitrated peptides was obtained by directly comparing SERS bands at 330-400cm(-1), allowing the rapid identification of PTN. Furthermore, without any pretreatments, the established method was successfully applied in the rapid in-situ dynamic monitoring of the mimic hemin-catalyzed PTN process in synthetic peptide, bovine serum albumin (BSA), and original human blood serum samples. The results indicated that the proposed approach could be a promising in-situ label-free tool for observing PTN process, which may be quite helpful to deeply understand the mechanism of post-translation modification.
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Two copper(II) complexes, 1 and 2, of two ligands, L1 and L2 [L1 = 2-{[anthracen-9-ylmethyl-(2-dimethylamino-ethyl)-amino]-methyl}-4,6-di-tert-butyl-phenol; L2 = 5-dimethylamino-naphthalene-1-sulfonic acid (3,5-di-tert-butyl-2-hydroxy-benzyl)-(2-dimethylamino-ethyl)-amide, were synthesized and characterized. In methanol solution, the quenched fluorescence intensity of the ligands in complexes 1 and 2 was found to be restored upon exposure to nitrogen dioxide. This is attributed to the reduction of the paramagnetic Cu(II) centre by nitrogen dioxide to diamagnetic Cu(I). The reduction was accompanied by simultaneous nitration in the phenol ring of the ligands.
Involvement of peroxynitrite (ONOO−) in inflammatory diseases has been implicated by detection of 3-nitrotyrosine, an allegedly characteristic protein oxidation
product, in various inflamed tissues. We show here that nitrite (NO2−), the primary metabolic end product of nitric oxide (NO·), can be oxidized by the heme peroxidases horseradish peroxidase, myeloperoxidase (MPO), and lactoperoxidase (LPO), in the
presence of hydrogen peroxide (H2O2), to most likely form NO·2, which can also contribute to tyrosine nitration during inflammatory processes. Phenolic nitration by MPO-catalyzed NO2− oxidation is only partially inhibited by chloride (Cl−), the presumed major physiological substrate for MPO. In fact, low concentrations of NO2− (2-10 μM) catalyze MPO-mediated oxidation of Cl−, indicated by increased chlorination of monochlorodimedon or 4-hydroxyphenylacetic acid, most likely via reduction of MPO
compound II. Peroxidase-catalyzed oxidation of NO2−, as indicated by phenolic nitration, was also observed in the presence of thiocyanate (SCN−), an alternative physiological substrate for mammalian peroxidases. Collectively, our results suggest that NO2−, at physiological or pathological levels, is a substrate for the mammalian peroxidases MPO and lactoperoxidase and that formation
of NO2· via peroxidase-catalyzed oxidation of NO2− may provide an additional pathway contributing to cytotoxicity or host defense associated with increased NO· production.
Nitrotyrosine formation is a hallmark of vascular inflammation, with polymorphonuclear neutrophil–derived (PMN-derived) and monocyte-derived myeloperoxidase (MPO) being shown to catalyze this posttranslational protein modification via oxidation of nitrite (NO2–) to nitrogen dioxide (NO2•). Herein, we show that MPO concentrates in the subendothelial matrix of vascular tissues by a transcytotic mechanism and serves as a catalyst of ECM protein tyrosine nitration. Purified MPO and MPO released by intraluminal degranulation of activated human PMNs avidly bound to aortic endothelial cell glycosaminoglycans in both cell monolayer and isolated vessel models. Cell-bound MPO rapidly transcytosed intact endothelium and colocalized abluminally with the ECM protein fibronectin. In the presence of the substrates hydrogen peroxide (H2O2) and NO2–, cell and vessel wall–associated MPO catalyzed nitration of ECM protein tyrosine residues, with fibronectin identified as a major target protein. Both heparin and the low–molecular weight heparin enoxaparin significantly inhibited MPO binding and protein nitrotyrosine (NO2Tyr) formation in both cultured endothelial cells and rat aortic tissues. MPO–/– mice treated with intraperitoneal zymosan had lower hepatic NO2Tyr/tyrosine ratios than did zymosan-treated wild-type mice. These data indicate that MPO significantly contributes to NO2Tyr formation in vivo. Moreover, transcytosis of MPO, occurring independently of leukocyte emigration, confers specificity to nitration of vascular matrix proteins.
Lipoxygenases are non-heme iron dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids. Using soybean lipoxygenase-1 as a model, we have shown that two classes of lipoxygenase inhibitors currently in development as potential antiinflammatory agents obtain a significant amount of their potency by reducing the lipoxygenase active-site iron from the active ferric state to the inactive ferrous state. It is not surprising that the members of the first of these classes, the 2-benzyl-1-naphthols, are reducing agents. The members of the second class, the N-alkyl-hydroxamic acids, were not anticipated to be sufficiently strong reducing agents to be oxidized by the lipoxygenase ferric center; that they are provides additional evidence for that iron having a high reduction potential. This brings to (at least) five the number of classes of lipoxygenase inhibitors that are capable of reducing the active-site ferric ion and suggests the generality of this approach in the rational design of lipoxygenase inhibitors.
Hemin, in the presence of 2-mercaptoethanol and oxygen, catalyzes the selective degradation of heme-binding proteins to small peptide fragments. Among the proteins examined, the heme-binding protein of rabbit serum (HBP-93) proved to be unusually sensitive. Myoglobin also exhibited considerable sensitivity whereas hemopexin and bovine serum albumin were only slightly susceptible to this degradative action of hemin. The reaction with HBP-93 depended upon coordination of the protein with hemin, was optimal at pH 6.5 and increased 4-fold as the temperature was elevated from 10 to 60 degrees C. The requirement for both oxygen and the reducing agent, 2-mercaptoethanol, and the partial protection of HBP-93 to degradation by catalase, superoxide dismutase, mannitol, and thiourea suggest the involvement of reduced oxygen species in the reaction. A possible role for the heme-mediated degradation of proteins in cell differentiation and other biological responses is discussed.
Phagocytic cytochrome b558 is a unique heme-containing enzyme, which catalyzes one electron reduction of molecular oxygen to produce a superoxide anion
with a six-coordinated heme iron. To clarify the mechanism of the superoxide production, we have analyzed oxidation-reduction
kinetics of cytochrome b558 purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b558 was rapidly reoxidized by O2 showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome
b558 with Na2S2O4 under anaerobic conditions. The single turnover rate for the reaction with O2 linearly depended on the O2 concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a
pKα of 9.7. Under complete anaerobic conditions, ferrous cytochrome b558 was oxidized by ferricyanide at a rate faster than by O2. The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b558 are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the
iron-O2 complex. These findings are most consistent with the electron transfer from the heme to O2 by an outer-sphere mechanism.
Iron-derived reactive oxygen species are implicated in the pathogenesis of various vascular disorders including atherosclerosis, vasculitis, and reperfusion injury. The present studies examine whether heme, when liganded to physiologically relevant proteins as in hemoglobin, can provide potentially damaging iron to intact endothelium. We demonstrate that reduced ferrohemoglobin, while relatively innocuous to cultured endothelial cells, when oxidized to ferrihemoglobin (methemoglobin), greatly amplifies oxidant (H2O2)-mediated endothelial-cell injury. Drawing upon our previous observation that free heme similarly primes endothelium for oxidant damage, we posited that methemoglobin, but not ferrohemoglobin, releases its hemes that can then be incorporated into endothelial cells. In support, cultured endothelial cells exposed to methemoglobin--in contrast to exposure to ferrohemoglobin, cytochrome c, or metmyoglobin--rapidly increased their heme oxygenase mRNA and enzyme activity, thereby supporting heme uptake; ferritin production was also markedly increased after such exposure, thus attesting to eventual incorporation of Fe. These cellular methemoglobin effects were inhibited by the heme-scavenging protein hemopexin and by haptoglobin or cyanide, agents that strengthen the liganding between heme and globin. If the endothelium is exposed to methemoglobin for a more prolonged period (16 hr), it accumulates large amounts of ferritin; concomitantly, and presumably associated with iron sequestration by this protein, the endothelium converts from hypersusceptible to hyperresistant to oxidative damage. We conclude that when oxidation of hemoglobin facilitates release of its heme groups, catalytically active iron is provided to neighboring tissue environments. The effect of this relinquished heme on the vasculature is determined both by extracellular factors--i.e., plasma proteins, such as haptoglobin and hemopexin--as well as intracellular factors, including heme oxygenase and ferritin. Acutely, if both extra- and intracellular defenses are overwhelmed, cellular toxicity arises; chronically, when ferritin is induced, resistance to oxidative injury may supervene.
Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.
The capabilities of stimulated neutrophils to initiate intraphagosomal and extracellular chlorination, nitration, and other oxidative reactions has been evaluated using a fluorescent particle and soluble phenolic compounds as target molecules. Neutrophils activated by the soluble stimulus, phorbol myristate acetate, both chlorinated fluorescein that was covalently attached to polyacrylamide microspheres and initiated tyrosine dimerization. When nitrite ion was present at millimolar concentration levels in the medium, nitration of the phenolic rings also occurred; the relative extent of nitration increased as the nitrite concentration was increased. Myeloperoxidase (MPO) also catalyzed nitration and chlorination of fluorescein and the fluorescein-conjugated particles in cell-free solutions; the relative nitration yields increased with increasing [NO2-]/[Cl-] ratios. Nitration did not involve intermediary formation of nitrating agents derived from reaction between MPO-generated HOCl and NO2- because this reaction also occurred in chloride-free media and direct addition of HOCl to solutions containing NO2- and fluorescein gave only chlorinated products. In marked contrast to these extracellular reactions, intraphagosomal nitration of the fluorescein-conjugated particles could not be detected (even at [NO2-] as high as 0.1 M), whereas chlorination of the probe was extensive. These data indicate that intraphagosomal aromatic nitration in neutrophils is negligible, although extracellular nitration of phenolic compounds by secreted MPO could occur at physiological concentration levels of NO2-.
Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. Heme serves as the prosthetic group of numerous hemoproteins (eg, hemoglobin, myoglobin, cytochromes, guanylate cyclase, and nitric oxide synthase) and plays an important role in controlling protein synthesis and cell differentiation. Cellular heme levels are tightly controlled; this is achieved by a fine balance between heme biosynthesis and catabolism by the enzyme heme oxygenase. On a per-cell basis, the rate of heme synthesis in the developing erythroid cells is at least 1 order of magnitude higher than in the liver, which is in turn the second most active heme producer in the organism. Differences in iron metabolism and in genes for 5-aminolevulinic acid synthase (ALA-S, the first enzyme in heme biosynthesis) are responsible for the differences in regulation and rates of heme synthesis in erythroid and nonerythroid cells. There are 2 different genes for ALA-S, one of which is expressed ubiquitously (ALA-S1), whereas the expression of the other (ALA-S2) is specific to erythroid cells. Because the 5'-untranslated region of the erythroid-specific ALA-S2 mRNA contains the iron-responsive element, a cis-acting sequence responsible for translational induction of erythroid ALA-S2 by iron, the availability of iron controls protoporphyrin IX levels in hemoglobin-synthesizing cells. In nonerythroid cells, the rate-limiting step of heme production is catalyzed by ALA-S1, whose synthesis is feedback-inhibited by heme. On the other hand, in erythroid cells, heme does not inhibit either the activity or the synthesis of ALA-S but does inhibit cellular iron acquisition from transferrin without affecting its utilization for heme synthesis. This negative feedback is likely to explain the mechanism by which the availability of transferrin iron limits heme synthesis rate. Moreover, in erythroid cells heme seems to enhance globin gene transcription, is essential for globin translation, and supplies the prosthetic group for hemoglobin assembly. Heme may also be involved in the expression of other erythroid-specific proteins. Furthermore, heme seems to play a role in regulating either transcription, translation, processing, assembly, or stability of hemoproteins in nonerythroid cells. Heme oxygenase, which catalyzes heme degradation, seems to be an important enzymatic antioxidant system, probably by providing biliverdin, which is an antioxidant agent.
Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.
Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.
The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys(75) and His(77) are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro(2) is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys(75) and His(77) is thought to cause the difference between the reduction and oxidation midpoint potentials.
Nitric oxide (NO), a simple free radical gas, elicits a surprisingly wide range of physiological and pathophysiological effects. NO interacts with soluble guanylate cyclase to evoke many of these effects. However, NO can also interact with molecular oxygen and superoxide radicals to produce reactive nitrogen species that can modify a number of macromolecules including proteins, lipids, and nucleic acids. NO can also interact directly with transition metals. Here, we have reviewed the non--3',5'-cyclic-guanosine-monophosphate-mediated effects of NO including modifications of proteins, lipids, and nucleic acids.
Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.
Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H(2)O(2): an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H(2)O(2) turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H(2)O(2) generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H(2)O(2) can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H(2)O(2) forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H(2)O(2) in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the beta-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H(2)O(2) from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H(2)O(2) metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.
The chemical origins of nitrated tyrosine residues (NT) formed in proteins during a variety of pathophysiological conditions remain controversial. Although numerous studies have concluded that NT is a signature for peroxynitrite (ONOO(-)) formation, other works suggest the primary involvement of peroxidases. Because metal homeostasis is often disrupted in conditions bearing NT, the role of metals as catalysts for protein nitration was examined. Cogeneration of nitric oxide (NO) and superoxide (O(2)(-)), from spermine/NO (2.7 microM/min) and xanthine oxidase (1-28 microM O(2)(-)/min), respectively, resulted in protein nitration only when these species were produced at approximately equivalent rates. Addition of ferriprotoporphyrin IX (hemin) to this system increased nitration over a broad range of O(2)(-) concentrations with respect to NO. Nitration in the presence of superoxide dismutase but not catalase suggested that ONOO(-) might not be obligatory to this process. Hemin-mediated NT formation required only the presence of NO(2)(-) and H(2)O(2), which are stable end-products of NO and O(2)(-) degradation. Ferrous, ferric, and cupric ions were also effective catalysts, indicating that nitration is mediated by species capable of Fenton-type chemistry. Although ONOO(-) can nitrate proteins, there are severe spatial and temporal constraints on this reaction. In contrast, accumulation of metals and NO(2)(-) subsequent to NO synthase activity can result in far less discriminate nitration in the presence of an H(2)O(2) source. Metal catalyzed nitration may account for the observed specificity of protein nitration seen under pathological conditions, suggesting a major role for translocated metals and the labilization of heme in NT formation.
The role of nitric oxide in cellular signaling in the past 22 years has become one of the most rapidly growing areas in biology with more than 20,000 publications to date. Nitric oxide is a gas and free radical with an unshared electron that can regulate an ever-growing list of biological processes. In many instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis from GTP. However, the list of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate its effect, while other reactions can lead to altered protein structure, function, and/or catalytic capacity. These diverse effects of nitric oxide that are either cyclic GMP dependent or independent can alter and regulate important physiological and biochemical events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an autacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique simple molecule with an array of signaling functions. However, as with any messenger molecule, there can be too little or too much of the substance and pathological events result. Some of the methods to regulate either nitric oxide formation, metabolism, or function have been in clinical use for more than a century as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870's. Current and future research with nitric oxide and cyclic GMP will undoubtedly expand the clinicians' therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide and cyclic GMP formation and metabolism. Such promise and expectations have obviously fueled the interests in these signaling molecules for a growing list of potential therapeutic applications.
On October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine and Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system. In contrast with the short research history of the enzymatic synthesis of NO, the introduction of nitrate-containing compounds for medicinal purposes marked its 150(th) anniversary in 1997. Glyceryl trinitrate (nitroglycerin; GTN) is the first compound of this category. Alfred Nobel (the founder of Nobel Prize) himself had suffered from angina pectoris and was prescribed nitroglycerin for his chest pain. Almost a century later, research in the NO field has dramatically extended and the role of NO in physiology and pathology has been extensively studied. The steady-state concentration and the biological effects of NO are critically determined not only by its rate of formation, but also by its rate of decomposition. Biotransformation of NO and its related N-oxides occurs via different metabolic routes within the body and presents another attractive field for our research as well as for the venture of drug discovery.
Hypochlorite oxidation of NO2- does not take place by oxygen atom transfer, but proceeds by Cl+ transfer from HOCl to NO2- to give NO2Cl as a reaction intermediate. The kinetics indicate that the subsequent decomposition of NO2Cl proceeds by two pathways: loss of Cl- to give NO2+ and reaction of NO2Cl with NO2- to form N2O4 and Cl-. At high Cl- and low OH- and NO2- concentrations the overall rate of NO3- formation is suppressed by Cl-. The relative reactivities for the reaction with NO2+ are OH- >> Cl- >> H2O. Although oxygen isotope experiments are consistent with a Cl+ transfer mechanism, the rate of exchange of oxygen between OCl- and H2O is relatively rapid (even at high pH in the absence of Cl-). We predict that the OCl-/H2O exchange rate in base will be independent of OH- concentration.
Publisher Summary This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.
Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.
In the absence of reductant substrates, and with excess H2O2, peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) shows the kinetic behaviour of a suicide inactivation, H2O2 being the suicide substrate. From the complex (compound I-H2O2), a competition is established between two catalytic pathways (the catalase pathway and the compound III-forming pathway), and the suicide inactivation pathway (formation of inactive enzyme). A kinetic analysis of this system allows us to obtain a value for the inactivation constant, ki = (3.92 +/- 0.06) x 10(-3) x s-1. Two partition ratios (r), defined as the number of turnovers given by one mol of enzyme before its inactivation, can be calculated: (a) one for the catalase pathway, rc = 449 +/- 47; (b) the other for the compound III-forming pathway, rCoIII = 2.00 +/- 0.07. Thus, the catalase activity of the enzyme and, also, the protective role of compound III against an H2O2-dependent peroxidase inactivation are both shown to be important.
The discovery of the enzymatic production of the superoxide (O2·⁻) radical and of the presence of superoxide dismutase (SOD) enzymes in aerobic cells led directly to the proposal that O2·⁻ is a major factor in oxygen toxicity and that SOD constitutes an important defense against it. Systems generating the O2·⁻ radical have been shown to have a number of damaging effects, some of which are summarized. The superoxide radical itself in organic solvents is a powerful base and nucleophile, which may have relevance to reactions taking place within the interior of cell membranes. In many cases, damage is decreased by addition not only of SOD but also of catalase, and it was proposed that O2·⁻ and H2O2 can combine together directly to generate the highly reactive hydroxyl radical, OH·. Indeed, damage is often decreased by the scavengers of this radical, such as mannitol, sodium formate, and thiourea. In such experiments, a range of scavengers should be used and it ought to be possible to correlate the degree of protection that they offer with the known rate constants for reaction of the scavengers with OH·. Several authors proposed that the salts of transition metals could catalyze the generation of hydroxyl radical, although most direct evidence for this has come from work with iron salts.
1. Peroxidases typically follow the reaction cycle: native enzyme-->compound I-->compound II-->native enzyme, in which the latter two steps involve hydrogen atom transfer from substrate to enzyme. 2. Exceptions involve (1) very facile, rapidly reacting reducing substrates that transfer an electron rather than a hydrogen atom, resulting in formation of a substrate pi-cation radical; (2) two two-electron transfer steps: native enzyme-->compound I-->native enzyme; and (3) compound III and the reduced form of the enzyme containing iron(II). 3. Prostaglandin H synthase is a peroxidase with some of the properties of a P450 in that compound I can abstract the hydrogen atom from a C-H bond. 4. The so-called cyclooxygenase and peroxidase activities of prostaglandin H synthase are intimately connected and, with the above exception, both are part of a conventional peroxidase cycle.
Ascorbate is catalytically oxidized by a coupled iron-ceruloplasmin system, the iron ions functioning as a red/ox cycling intermediate between ceruloplasmin and ascorbate. Serum albumin, an iron binding compound, was found to stimulate the ascorbate oxidation rate. It is proposed that ferrous ions react more rapidly with ceruloplasmin when they are bound to albumin. A K
m value of 39 μ
m was estimated for Fe2+-albumin. Citrate and urate inhibit the iron-ceruloplasmin-dependent ascorbate oxidation by chelating ferric ions. In the presence of albumin only citrate reduced the oxidation rate, the observation suggesting the following order of iron binding ability: citrate > albumin > urate. Physiological aspects of the results have been discussed.
Iron and hydrogen peroxide are capable of oxidizing a wide range of substrates and causing biological damage. The reaction, referred to as the Fenton reaction, is complex and capable of generating both hydroxyl radicals and higher oxidation states of the iron. The mechanism and how it is affected by different chelators, and the interpretation of results obtained in biological systems, are discussed.
The SR Ca-ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca-ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O2. These data suggest the vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue implies an age-associated increase in susceptibility to oxidation by this species.
Production of reactive species has been associated with tissue injury in diverse human disorders and experimental models of disease. Peroxynitrite is a strong oxidant with multiple pathways of reactivity. One protein modification reaction that may be specific to peroxynitrite is the nitration of the ortho position of tyrosine residues and nitrotyrosine has been used as a marker for peroxynitrite-mediated oxidative stress. Nitrotyrosine was formed when peroxynitrite was reacted at physiological pH with fatty acid-free bovine serum albumin or with human plasma proteins. Nitrotyrosine was not formed when proteins were incubated with nitric oxide, nitrogen dioxide, or nitric oxide plus hydrogen peroxide in the presence of ferrous iron or ferrihorseradish peroxidase. Low-molecular-weight molecules such as uric acid, ascorbate, and sulfhydryls inhibited protein tyrosine nitration in the absence of bicarbonate. Addition of bicarbonate catalytically enhanced the yield of nitration and overcame the inhibition of these antioxidants. Bicarbonate/CO2 enhanced the yield of protein nitrotyrosine in a concentration-dependent manner. Catalysis of nitration is achieved by the interaction of CO2 with the peroxynitrite anion. A mechanism is proposed involving an ONOO(O)CO- intermediate, which readily nitrates tyrosine residues in a non-radical-dependent manner. Thus, peroxynitrite nitrates tyrosine residues by a mechanism that is catalyzed by CO2 under normal physiological conditions.
Nitric oxide signaling during the past two decades has been one of the most rapidly growing areas in biology. This simple free radical gas with an unshared electron can regulate an ever-growing list of biological processes. In most instances, nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, effects of nitric oxide that are independent of cyclic GMP are also growing at a rapid rate. Nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids, and other reactive species. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication, and signaling. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism, or function have been used therapeutically for more than a century, as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.
Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.
Major advances have been made in our understanding of cytochrome c oxidase owing to continued crystallographic work on important intermediates. This, together with a wealth of data derived from selective mutations and sophisticated spectroscopic probes, has provided significant new insights into oxidase dioxygen chemistry and proton pumping activities. Recent advances have also been made for nitric oxide synthase, owing to the crystal structure determination of the heme domain for two of three nitric oxide synthase isoforms.
The presence of nitrotyrosine in the kidney has been associated with several pathological conditions. In the present study, we investigated nitrotyrosine formation in rat kidney after animals received endotoxin for 24 h. With lipopolysaccharide (LPS) treatment, immunohistochemical data demonstrated intense nitrotyrosine staining throughout the kidney. In spite of marked nitrotyrosine formation, the architectural appearance of tubules, glomeruli, and capillaries remained intact when examined by reticulin staining. Our data suggested that the marked staining of nitrotyrosine in proximal tubular epithelial cells was in the subapical compartment where the endocytic lysosomal apparatus is located. Thus a large portion of nitrotyrosine may come from the hydrolysis of nitrated proteins that are reabsorbed by the proximal tubule during the LPS treatment. We also found the colocalization of nitric oxide synthase (NOS-1) and nitrotyrosine within the macula densa of LPS-treated rats by using a double fluorescence staining method. In renal arterial vessels, vascular endothelial cells were more strongly stained for nitrotyrosine than vascular smooth muscle cells. Control animals without LPS treatment showed much less renal staining for nitrotyrosine. The general distribution of nitrotyrosine staining in control rat renal cortex is in the proximal and convoluted tubules, whereas the endothelial cells of vasa recta are major areas of nitrotyrosine staining in inner medulla. The renal distribution of nitrotyrosine in control and LPS-treated animals suggests that protein nitration may participate in renal regulation and injury in ways that are yet to be defined.
The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.
The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15 degrees C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8x10(7) M(-1) s(-1) and 1.4x10(7) M(-1) s(-1), respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2x10(5) M(-1) s(-1) and 2.7x10(6) M(-1) s(-1). Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6. 6x10(4) M(-1) s(-1) at pH 7 and 15 degrees C). Both reactions are controlled by the same ionizable group (average pK(a) of about 4.0) which has to be in its conjugated base form for reaction.
Nitric oxide (NO) possesses potent anti-inflammatory properties; however, an over-production of NO will promote inflammation and induce cell and tissue dysfunction. Thus, the ability to precisely regulate NO production could prove beneficial in controlling damage. In this study, advantage was taken of the well characterized inflammatory response caused by an intestinal parasite, Trichinella spiralis, to study the relationship between intestinal inflammation and the regulation of nitric oxide synthase-type 2 (NOS-2) expression. Our study revealed that a specific gut inflammatory reaction results in inhibition of NOS-2 expression. Characteristics of this inhibition are: 1) local jejunal inflammation induced by T. spiralis systemically inhibits NOS-2 gene transcription, protein expression, and enzyme activity; 2) the inhibition blunts endotoxin-stimulated NOS-2 expression; 3) the inhibition does not extend to the expression of other isoforms of NOS, to paxillin, a housekeeper protein, or to cyclooxygenase-2, another protein induced by proinflammatory cytokines; 4) the inhibition is unlikely related to the formation of specific anti-parasite antibodies; and 5) the inhibition may involve substances other than stress-induced corticosteroids. Elucidation of such potent endogenous NOS-2 down-regulatory mechanisms could lead to the development of new strategies for the therapy of inflammatory conditions characterized by the overproduction of NO.
Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.
We recently described that horseradish peroxidase (HRP) and myeloperoxidase (MPO) catalyze the oxidation of melatonin, forming the respective indole ring-opening product N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) (Biochem. Biophys. Res. Commun. 279, 657-662, 2001). Although the classic peroxidatic enzyme cycle is expected to participate in the oxidation of melatonin, the requirement of a low HRP:H(2)O(2) ratio suggested that other enzyme paths might also be operative. Here we followed the formation of AFMK under two experimental conditions: predominance of HRP compounds I and II or presence of compound III. Although the consumption of substrate is comparable under both conditions, AFMK is formed in significant amounts only when compound III predominates during the reaction. Using tryptophan as substrate, N- formyl-kynurenine is formed in the presence of compound III. Both, melatonin and tryptophan efficiently prevents the formation of p-670, the inactive form of HRP. Since superoxide dismutase (SOD) inhibits the production of AFMK, we proposed that compound III acts as a source of O(-*)(2) or participates directly in the reaction, as in the case of enzyme indoleamine 2,3-dioxygenase.
The eosinophilic inflammatory response in asthma is associated with protein nitration, detected as immunostaining for 3-nitrotyrosine (3NT). As the presence of 3NT is strongly correlated with upregulation of the inducible form of nitric oxide synthase (NOS II), it has been hypothesized that 3NT formation results from the action of peroxynitrite (ONOO-), a highly reactive NO derivative produced from the reaction of molecular NO and O2-. However, recent observations have suggested that the action of peroxidases, including eosinophil peroxidase (EPO), may be responsible for protein nitration. In this study, we used murine models of allergic asthma to address the relative contribution of EPO and NOS II to protein nitration. We studied EPO-deficient New Zealand White (NZW) mice, which were sensitized and challenged intranasally with ovalbumin (OVA). Despite comparable levels of eosinophilia, NO, and superoxide production, NZW mice exhibited markedly decreased 3NT staining around the airways after OVA challenge when compared with two other strains (A/J and C57BL/6J). Immunocytochemical analysis of bronchoalveolar lavage (BAL) cells and lung sections suggested that 3NT staining was largely confined to eosinophils. This was confirmed by Western Blot analysis of proteins from different subsets of BAL cells that demonstrated a marked decrease in 3NT formation in eosinophils from NZW mice. These results contrast with those obtained in OVA-sensitized and -challenged NOS II deficient mice, which despite decreased NO production, exhibited similar 3NT staining in the airways after OVA challenge as in wild-type control mice. In this model, protein nitration was thus not a function of NO production by NOS II. We conclude that in the mouse, 3NT formation after specific allergen challenge is dependent on EPO activity, particularly in eosinophils themselves. In contrast, 3NT formation is not driven by upregulation of NOS II expression in this model and does not appear to depend on increases in the level of NO production.
Oxidation of the anticancer anthracyclines doxorubicin (DXR) and daunorubicin (DNR) by lactoperoxidase(LPO)/H(2)O(2) and horseradish peroxidase(HRP)/H(2)O(2) systems in the presence and absence of nitrite (NO(2)(-)) has been investigated using spectrophotometric and EPR techniques. We report that LPO/H(2)O(2)/NO(2)(-) causes rapid and irreversible loss of anthracyclines' absorption bands, suggesting oxidative degradation of their chromophores. Both the initial rate and the extent of oxidation are dependent on both NO(2)(-) concentration and pH. The initial rate decreases when the pH is changed from 7 to 5, and the reaction virtually stops at pH 5. Oxidation of a model hydroquinone compound, 2,5-di-tert-butylhydroquinone, by LPO/H(2)O(2) is also dependent on NO(2)(-); however, in contrast to DNR and DXR, this oxidation is most efficient at pH 5, indicating that LPO/H(2)O(2)/NO(2)(-) is capable of efficiently oxidizing simple hydroquinones even in the neutral form. Oxidation of anthracyclines by HRP/H(2)O(2)/NO(2)(-) is substantially less efficient relative to that by LPO/H(2)O(2)/NO(2)(-) at either pH 5 or pH 7, most likely due to the lower rate of NO(2)(-) metabolism by HRP/H(2)O(2). EPR measurements show that interaction of anthracyclines and 2,5-di-tert-butylhydroquinone with LPO/H(2)O(2)/NO(2)(-) generates the corresponding semiquinone radicals presumably via one-electron oxidation of their hydroquinone moieties. The possible role of the (*)NO(2) radical, a putative LPO metabolite of NO(2)(-), in oxidation of these compounds is discussed. Because in vivo the anthracyclines may co-localize with peroxidases, H(2)O(2), and NO(2)(-) in tissues, their oxidation via the proposed mechanism is likely. These observations reveal a novel, peroxidase- and nitrite-dependent mechanism for the oxidative transformation of the anticancer anthracyclines, which may be pertinent to their biological activities in vivo.
The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.
Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.
Iron is an essential metal for most biological organisms. However, if not tightly controlled, iron can mediate the deleterious oxidation of biomolecules. This review focuses on the current understanding of the role of iron in the deleterious oxidation of various biomolecules, including DNA, protein, lipid, and small molecules, e.g., ascorbate and biogenic amines. The effect of chelation on the reactivity of iron is also addressed, in addition to iron-associated toxicities. The roles of the iron storage protein ferritin as both a source of iron for iron-mediated oxidations and as a mechanism to safely store iron in cells is also addressed.
Nitrogen dioxide and carbonate radical anion have received sporadic attention thus far from biological investigators. However, accumulating data on the biochemical reactions of nitric oxide and its derived oxidants suggest that these radicals may play a role in various pathophysiological processes. These potential roles are also indicated by recent studies on the high efficiency of urate and nitroxides in protecting cells and whole animals against the injury associated with conditions of excessive nitric oxide production. The high protective effects of these antioxidants are incompletely defined at the mechanistic level but some of them can be explained by their efficiency in scavenging peroxynitrite-derived radicals, particularly nitrogen dioxide and carbonate radical anion. In this review, we provide a framework for this hypothesis and discuss the potential sources and properties of these radicals that are likely to become increasingly recognized as important mediators of biological processes.
Biological systems rely on heme-proteins to carry out a number of basic functions essential for their survival. Hemes, or iron-porphyrin complexes, are the versatile and ubiquitous active centers of these proteins. In the past decade, discovery of new heme-proteins, together with functional and structural research, provided a wealth of information on these diverse and biologically important molecules. Structure determination work has shown that nature has used a variety of different scaffolds and architectures to bind heme and modulate functions such as redox properties. Structural data have also provided insights into the heme-linked protein conformational changes required in many regulatory heme-proteins. Remarkable efforts have been made towards the understanding of factors governing redox potentials. Site-directed mutagenesis studies and theoretical calculations on heme environments investigated the roles of hydrophobic and electrostatic residues, and analyzed the effect of heme solvent accessibility. This review focuses on the structure-function relationships underlying the association of heme in signaling and iron metabolism proteins. In addition, an account is given about molecular features affecting heme's redox properties; this briefly revisits previous conclusions in the light of some more recent reports.
Iron is an essential nutrient for the growth, development, and long-term survival of most organisms. High tissue iron concentrations have been associated with the development and progression of several pathological conditions, including certain cancers, liver and heart disease, diabetes, hormonal abnormalities, and immune system dysfunctions. In this review we discuss the relevance of iron toxicity on free radical-mediated tissue damage, and how iron interactions with nutrient antioxidants and other metals can affect the extent of oxidative damage to different biomolecules. It can be concluded that the ingestion of antioxidant rich foods may prevent or delay primary and secondary effects associated with iron overload-related diseases.
On October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine and Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system. In contrast with the short research history of the enzymatic synthesis of NO, the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in 1997. Glyceryl trinitrate (nitroglycerin; GTN) is the first compound of this category. Alfred Nobel (the founder of Nobel Prize) himself had suffered from angina pectoris and was prescribed nitroglycerin for his chest pain. Almost a century later, research in the NO field has dramatically extended and the role of NO in physiology and pathology has been extensively studied. The steady-state concentration and the biological effects of NO are critically determined not only by its rate of formation, but also by its rate of decomposition. Biotransformation of NO and its related N-oxides occurs via different metabolic routes within the body and presents another attractive field for our research as well as for the venture of drug discovery.
The role of nitric oxide in cellular signaling in the past 22 years has become one of the most rapidly growing areas in biology with more than 20,000 publications to date. Nitric oxide is a gas and free radical with an unshared electron that can regulate an ever-growing list of biological processes. In many instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis from GTP. However, the list of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate its effect, while other reactions can lead to altered protein structure, function, and/or catalytic capacity. These diverse effects of nitric oxide that are either cyclic GMP dependent or independent can alter and regulate important physiological and biochemical events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an autacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique simple molecule with an array of signaling functions. However, as with any messenger molecule, there can be too little or too much of the substance and pathological events result. Some of the methods to regulate either nitric oxide formation, metabolism, or function have been in clinical use for more than a century as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870's. Current and future research with nitric oxide and cyclic GMP will undoubtedly expand the clinicians' therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide and cyclic GMP formation and metabolism. Such promise and expectations have obviously fueled the interests in these signaling molecules for a growing list of potential therapeutic applications.