Fig 4 - uploaded by Dorival Martins
Content may be subject to copyright.
Ccp1 accumulates in the mitochondria of cta1Δ yeast. (A) Immunoblot analysis of denucleated (S2) and mitochondrial (P10) fractions isolated from wild-type (WT), cta1Δ, and ctt1Δ cells. (B) The Ccp1 signals for the P10 fractions in A were quantified and normalized to the porin signal (mitochondrial outer membrane marker) for 2-d (blue bars) and 7-d (red bars) cells. (C) Specific CCP activity (μmol·min −1 ·mg −1 total protein) of the P10 fractions in B ratioed by their Ccp1 protein levels to give the relative amount of active CCP remaining in mitochondria. Results in A are representative of three independent cultures (n = 3) and averages ± SD are plotted in B and C.
Source publication
Significance
We provide to our knowledge the first in vivo and in vitro evidence for H 2 O 2 -triggered heme transfer between proteins. Specifically, H 2 O 2 binds to and labilizes cytochrome c peroxidase (Ccp1)’s heme by oxidizing the proximal Fe ligand (His175), which activates Ccp1 to transfer its heme to apoCta1, and apoCcp1 subsequently escape...
Citations
... Furthermore, cell viability assays demonstrated that the deletion mutants displayed reduced tolerance to H 2 O 2 compared to the wild type (Fig. 4B). Interestingly, unlike ScCta1 in S. cerevisiae, which primarily clears cytosolic ROS, and ScCtt1, which deals with oxidative stress from external sources (29)(30)(31), the catalase CnCat in GXAS-CN plays a dominant role in combating H 2 O 2 -induced oxidative stress, as supported by RNA-seq data and the deletion mutant experiments. ...
Yeast cells involved in fermentation processes face various stressors that disrupt redox homeostasis and cause cellular damage, making the study of oxidative stress mechanisms crucial. In this investigation, we isolated a resilient yeast strain, Candida nivariensis GXAS-CN, capable of thriving in the presence of high concentrations of H 2 O 2 . Transcriptomic analysis revealed the up-regulation of multiple antioxidant genes in response to oxidative stress. Deletion of the catalase gene Cncat significantly impacted H 2 O 2 -induced oxidative stress. Enzymatic analysis of recombinant Cn Cat highlighted its highly efficient catalase activity and its essential role in mitigating H 2 O 2 . Furthermore, over-expression of Cn Cat in Saccharomyces cerevisiae improved oxidative resistance by reducing intracellular ROS accumulation. The presence of multiple stress-responsive transcription factor binding sites at the promoters of antioxidative genes indicates their regulation by different transcription factors. These findings demonstrate the potential of utilizing the remarkably tolerant C. nivariensis GXAS-CN or enhancing the resistance of S. cerevisiae to improve the efficiency and cost-effectiveness of industrial fermentation processes.
IMPORTANCE
Enduring oxidative stress is a crucial trait for fermentation strains. The importance of this research is its capacity to advance industrial fermentation processes. Through an in-depth examination of the mechanisms behind the remarkable H 2 O 2 resistance in Candida nivariensis GXAS-CN and the successful genetic manipulation of this strain, we open the door to harnessing the potential of the catalase Cn Cat for enhancing the oxidative stress resistance and performance of yeast strains. This pioneering achievement creates avenues for fine-tuning yeast strains for precise industrial applications, ultimately leading to more efficient and cost-effective biotechnological processes.
... 10 In yeast cytochrome c peroxidase (CcP) hole hopping pathways operate to help protect, under high H 2 O 2 levels, from irreversible oxidation of the distal His-heme ligand, which results in loss of the heme and formation of the apo-protein. 11,12 Furthermore, hole hopping pathways can have functional implications. For example, the formation of stable, residue specic radical sites, can serve as a functional adaptation in the enzyme, through enhancing an electron-transfer pathway for substrate oxidation as is the case for the Trp p-radical cation, Trp191c + in CcP, [13][14][15][16] or be directly involved in the oxidation of a substrate through formation of neutral surface Tyr or Trp radicals in lignin and versatile peroxidases (LiP and VP, respectively). ...
In heme enzymes, such as members of the dye-decolorising peroxidase (DyP) family, the formation of the highly oxidising catalytic Fe(iv)-oxo intermediates following reaction with hydrogen peroxide can lead to free radical migration (hole hopping) from the heme to form cationic tyrosine and/or tryptophan radicals. These species are highly oxidising (∼1 V vs. NHE) and under certain circumstances can catalyse the oxidation of organic substrates. Factors that govern which specific tyrosine or tryptophan the free radical migrates to in heme enzymes are not well understood, although in the case of tyrosyl radical formation the nearby proximity of a proton acceptor is a recognised facilitating factor. By using an A-type member of the DyP family (DtpAa) as an exemplar, we combine protein engineering, X-ray crystallography, hole-hopping calculations, EPR spectroscopy and kinetic modelling to provide compelling new insights into the control of radical migration pathways following reaction of the heme with hydrogen peroxide. We demonstrate that the presence of a tryptophan/tyrosine dyad motif displaying a T-shaped orientation of aromatic rings on the proximal side of the heme dominates the radical migration landscape in wild-type DtpAa and continues to do so following the rational engineering into DtpAa of a previously identified radical migration pathway in an A-type homolog on the distal side of the heme. Only on disrupting the proximal dyad, through removal of an oxygen atom, does the radical migration pathway then switch to the engineered distal pathway to form the desired tyrosyl radical. Implications for protein design and biocatalysis are discussed.
... Heme (or haem) is a coordination complex of ferrous ion and porphyrin that is a central component of hemoglobin, where it acts as a carrier of oxygen. However, because of its intrinsic electrochemical properties, this structure is also found in other enzymes, including cytochromes and some types of peroxidase [73][74][75][76][77]. Because heme includes a porphyrin structure, its optical features are also similar to those of other porphyrin-containing molecules. ...
Photoimaging and phototherapy have become major platforms for the diagnosis and treatment of various health complications. These applications require a photosensitizer (PS) that is capable of absorbing light from a source and converting it into other energy forms for detection and therapy. While synthetic inorganic materials such as quantum dots and gold nanorods have been widely explored for their medical diagnosis and photodynamic (PDT) and photothermal (PTT) therapy capabilities, translation of these technologies has lagged, primarily owing to potential cytotoxicity and immunogenicity issues. Of the various photoreactive molecules, the naturally occurring endogenous compound heme, a constituent of red blood cells, and its derivatives, porphyrin, biliverdin and bilirubin, have shown immense potential as noteworthy candidates for clinically translatable photoreactive agents, as evidenced by previous reports. While porphyrin-based photomedicines have attracted significant attention and are well documented, research on photomedicines based on two other heme-derived compounds, biliverdin and bilirubin, has been relatively lacking. In this review, we summarize the unique photoproperties of heme-derived compounds and outline recent efforts to use them in biomedical imaging and phototherapy applications.
... Although LH is largely oxidized in HEK293 cells, it may vary between different cell types and be highly dynamic and responsive to various stimuli. The Fe(III)/Fe(II) redox couple is linked to a number of factors, including access to certain cellular reductants or oxidants (98), ligand binding to the heme iron center, e.g. CO or NO (96), and allosteric protein conformational changes (99). ...
Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and the constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.
... This suggests that reducing heme demand through deletion of cyt2 yields more available heme for the production of heterologous heme peroxidases. Greater heme availability may also allow greater production of native peroxide-detoxifying enzymes such as cytochrome c peroxidase and catalase 44 , mitigating possible oxidative effects of heterologous peroxidase expression in the secretory pathway. This deletion however results in an inability for the strain to grow on non-fermentable carbon sources, meaning that while cellular productivity is increased, cell density under inducing, proteinproducing conditions is limited. ...
Efficient, large-scale heterologous production of enzymes is a crucial component of the biomass valorization industry. Whereas cellulose utilization has been successful in applications such as bioethanol, its counterpart lignin remains significantly underutilized despite being an abundant potential source of aromatic commodity chemicals. Fungal lignin-degrading heme peroxidases are thought to be the major agents responsible for lignin depolymerization in nature, but their large-scale production remains inaccessible due to the genetic intractability of basidiomycete fungi and the challenges in the heterologous production of these enzymes. In this study, we employ a strain engineering approach based on functional genomics to identify mutants of the model yeast Saccharomyces cerevisiae with enhanced heterologous production of lignin-degrading heme peroxidases. We show that our screening method coupling an activity-based readout with fluorescence-assisted cell sorting enables identification of two single null mutants of S. cerevisiae, pmt2 and cyt2, with up to 11-fold improved secretion of a versatile peroxidase from the lignin-degrading fungus Pleurotus eryngii. We demonstrate that the double deletion strain pmt2cyt2 displays positive epistasis, improving and even enabling production of members from all three classes of lignin-degrading fungal peroxidases. We anticipate that these mutant strains will be broadly applicable for improved heterologous production of this biotechnologically important class of enzymes.
... Although CCP peroxidase activity has been exhaustively investigated in vitro, recent in vivo evidence points to an important H 2 O 2 sensing role for the enzyme in yeast. 76 Yeast CCP has unusually high Trp (2.38%) and Tyr (4.76%) content (compared to UniProtKB/Swiss-Prot database averages), forming an extensive network (contact distance < 10Å) connected to the heme (PDB ID 2CYP, Fig. 6). 77 Exposure of CCP to excess H 2 O 2 in the absence of Fe 2+ -cyt c leads to extensive oxidation of Trp and Met residues, as well as Tyr crosslinking to form dityrosine. 78,79 Glutathione (GSH) inhibits oxidation at most of these residues, consistent with the presence of protective chains that direct holes away from the heme toward surface residues for scavenging by GSH. ...
Electrons can tunnel through proteins in microseconds with a modest release of free energy over distances in the 15 to 20 Å range. To span greater distances, or to move faster, multiple charge transfers (hops) are required. When one of the reactants is a strong oxidant, it is convenient to consider the movement of a positively charged “hole” in a direction opposite to that of the electron. Hole hopping along chains of tryptophan (Trp) and tyrosine (Tyr) residues is a critical function in several metalloenzymes that generate high-potential intermediates by reactions with O₂ or H₂O₂, or by activation with visible light. Examination of the protein structural database revealed that Tyr/Trp chains are common protein structural elements, particularly among enzymes that react with O₂ and H₂O₂. In many cases these chains may serve a protective role in metalloenzymes by deactivating high-potential reactive intermediates formed in uncoupled catalytic turnover.
... Although LH is largely oxidized in HEK293 cells, it may vary between different cell types and be highly dynamic and responsive to various stimuli. The Fe(III)/Fe(II) redox couple is linked to a number of factors, including access to certain cellular reductants or oxidants (94), ligand binding to the heme iron center, e.g. CO or NO (92), and allosteric protein conformational changes (95). ...
Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms: inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, and various HO-2 mutant alleles, we found that endogenous heme is too limiting to support HO-2 catalyzed heme degradation. Rather, we discovered that a novel role for HO-2 is to bind and buffer labile heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor in control of heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity by enzymatically degrading it. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.
... The second putative protein is mitochondrial cytochrome c peroxidase with measured mass $40 kDa (theoretical mass 40348.3 kDa, Table 1), a haeme oxidoreductase which functions as degrading ROS in mitochondria, thus being involved in the response to oxidative stress [33,34]. This enzyme, determined from Band 4, is localized in the mitochondrial intermembrane space and its main biological function is thought to be reduction of H 2 O 2 generated during aerobic respiration [35]. ...
... Our result confirms the idea of Martins et al. [34] that the peroxidase activity from cytochrome c is autonomous and the enzyme also plays a role in sensing hydrogen peroxide and regulation of the catalase activity. Kathiresan et al. [33] assume in their recent studies that CCP1 behaviour reminds more of a mitochondrial H 2 O 2 sensor rather than of a H 2 O 2 detoxifying catalytic protein. ...
The focus of the present study is to determine proteins responsible for the oxidative and toxic stress response in proliferating and stationary phase (G0) cultures. Therefore, the yeast Saccharomyces cerevisiae was treated with oxidative and drug compounds (H2O2, menadione, zeocin, and ibuprofen) in both phases. These substances were chosen to determine the redox status of the yeast. S. cerevisiae appeared to employ different strategies to ensure their antioxidant defence metabolism. Analysis, including sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry, was used in the search. The proteins were identified by SDS-PAGE, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry analysis, and Mascot database-fingerprint. The final step was determination of protein profiles of yeast S. cerevisiae in proliferating (M) and stationary phase (G0). Seven bands were determined and the corresponding proteins were proposed: cytochrome c peroxidase, glutathione S-transferase omega-like, NAPDH-dependent diflavin reductase, DNA replication fork-blocking protein, putative aryl alcohol dehydrogenase, AP-1-like transcription factor YAP5, GTP-binding protein. All putative proteins coincide with the literature database. A typical example of such an adaptation mechanism in the defence against oxidative damage is the synthesis of several glutathione and thioredoxin peroxidases in the yeast cell. A deeper investigation of the conserved mechanisms responsible for entry into, survival, and exit from quiescence in higher eukaryotes will help the development of new anticancer therapies, the study in the process of ageing and neurodegenerative diseases.
... However, it is possible that hydrogen peroxide could interact with heme directly to impair the response of the sensor (75). Additionally, increased ROS due to vacuolar and mitochondrial dysfunction could stimulate the expression of heme-containing proteins (e.g., catalase and peroxidases) to potentially drive heme into a protein-bound form that is no longer detectable by the sensor (83). Further support for an impact of ROS and oxidative stress comes from our demonstration that loss of mitochondrial Sod2 resulted in reduced cytosolic heme and impaired growth on hemin as an iron source. ...
Invasive fungal diseases are increasing in frequency, and new drug targets and antifungal drugs are needed to bolster therapy. The mechanisms by which pathogens obtain critical nutrients such as iron from heme during host colonization represent a promising target for therapy. In this study, we employed a fluorescent heme sensor to investigate heme homeostasis in Cryptococcus neoformans . We demonstrated that endocytosis is a key aspect of heme acquisition and that vacuolar and mitochondrial functions are important in regulating the pool of available heme in cells. Stress generated by oxidative conditions impacts the heme pool, as do the drugs artemisinin and metformin; these drugs have heme-related activities and are in clinical use for malaria and diabetes, respectively. Overall, our study provides insights into mechanisms of fungal heme acquisition and demonstrates the utility of the heme sensor for drug characterization in support of new therapies for fungal diseases.
... Recently, CcP was reported also to work as a mitochondrial H 2 O 2 sensor [28,29] and to be involved in haem storage. [30] The encounter state between Cc and CcP can be described using exclusively electrostatic interactions, and paramagnetic NMR experiments revealed that Cc samples just 15 % of the CcP surface to find the binding site. [31] Furthermore, PRE experiments showed that the encounter complex between Cc and CcP is populated for 30 % of the time, while the stereospecific, crystallographic complex, [32] has 70 % occupancy. ...
We present a novel approach to study transient protein‐protein complexes with standard, 9 GHz, and high‐field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso‐1‐cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1‐oxyl‐2,2,5,5‐tetramethyl‐Δ3‐pyrroline‐3‐methyl)‐methanethiosulfonate] attached at position 81 of Cc (SL−Cc). A dissociation constant KD of 20±4×10⁻⁶ M (EPR and NMR) and an equal amount of stereo‐specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc‐interaction surface, with the stereo‐specific complex.
