Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Exploring the structure function relationship of heme peroxidases: Molecular dynamics study on cytochrome c peroxidase variants.

Cytochrome c peroxidase (Ccp1) is a mitochondrial heme-containing enzyme that has served for decades as a chemical model to explore the structure function relationship of heme enzymes. Unveiling the impact of its heme pocket residues on the structural behavior, the non-covalent interactions and consequently its peroxidase activity has been a matter of increasing interest. To further probe these roles, we conducted intensive all-atom molecular dynamics simulations on WT and nineteen in-silico generated Ccp1 variants followed by a detailed structural and energetic analysis of H2O2 binding and pairwise interactions. Different structural analysis including RMSD, RMSF, radius of gyration and the number of Hydrogen bonds clearly demonstrate that none of the studied mutants induce a significant structural change relative to the WT behavior. In an excellent agreement with experimental observations, the structural change induced by all the studied mutant systems is found to be very localized only to their surrounding environment. The determined interaction energies between residues and Gibbs binding energies for the WT Ccp1 and the nineteen variants, helped to identify the precise effect of each mutated residues on both the binding of H2O2 and the non-covalent interaction and thus the overall peroxidase activity. The roles of surrounding residues in adopting unique distinctive electronic feature by Ccp1 has been discerned. Our valuable findings have clarified the functions of various residues in Ccp1 and thereby provided novel atomistic insights into its function. Overall, due to the conserved residues of the heme-pocket amongst various peroxidases, the obtained remarks in this work are highly valuable.

Improved Metabolic Pathways of Glycolysis, Glycogen Synthesis, the Urea Cycle, and Cytochrome Peroxidase Oxidative Reabsorption in a Miniature Bioreactor.

BACKGROUND/AIMS: Bioreactor-based bioartificial liver support systems have had limited success in a translational setting and at preclinical stages. None of the existing systems monitor the metabolic pathways of glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption. Herein, we designed a bioreactor that mimics the human liver microenvironment in vivo and monitors different hepatic metabolic pathways in order to help establish in vitro culture conditions for improved glycolysis, glycogen synthesis, the urea cycle, cytochrome peroxidase oxidative reabsorption and improved hepatic functions in a miniature bioartificial liver. An abnormality in such pathways negatively influences survivability and hepatic functions, including spontaneous liver regeneration. METHODS: We investigated the metabolic functions of primary mouse adult hepatocytes cultured in a three-dimensional configuration under direct oxygenation conditions (5%, 10%, 20%, and 40% O2) for 14 days in the bioreactor. We analyzed the expression of the genes of hepatic metabolic pathways, such as glycolysis (glucokinase, phosphofructokinase, and pyruvate kinase), glycogen synthesis (glycogen synthetase, UTP glucose-1-phosphate uridylylisomerase, phosphoglucomutase, and glycogen phosphorylase), the urea cycle (arginase, ornithine carbomoyltransferase, fumarate hydratase), oxidative reabsorption (peroxidase), and cytochrome peroxides (catalase and superoxide dismutase), and compared it with the level in vivo. The metabolic mini-map was used to represent the above-mentioned metabolic genes. RESULTS: Increased urea secretion under normoxia and hyperoxia conditions (20% and 40% O2, respectively) was observed, while albumin secretion was decreased in hyperoxic cultures. Lactate formation was up to 15 mg/L-g/h-h/106 cells, 2 mg/L-g/h-h/106 cells, and 0.2 mg/L-c/h-h/106 cells in 5%, 20%, and 40% O2 conditions, respectively while glucose consumption was enhanced under hypoxic conditions (5% and 10% O2). Cellular membrane integrity was estimated by lactate dehydrogenase assay and was found to be negligible in only 20% and 40% O2 conditions. The expression of the phase II enzyme UDP-glucuronosyltransferase was only upregulated in 20% oxygenation. CONCLUSION: Taken together, 20% O2 was found to be an optimal condition for the long-term culture (up to 14 days) of hepatocytes that promoted the expression of genes in metabolic pathways such as glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption, and improved hepatic functions in a miniature bioreactor for bioartificial liver construction.

Why Do Most Aromatics Fail to Support Hole Hopping in the Cytochrome c Peroxidase-Cytochrome c Complex?

Electron transport through aromatic species (especially tryptophan and tyrosine) plays a central role in water splitting, redox signaling, oxidative damage protection, and bioenergetics. The cytochrome c peroxidase (CcP)-cytochrome c (Cc) complex (CcP:Cc) is used widely to study interprotein electron transfer (ET) mechanisms. Tryptophan 191 (Trp191) of CcP supports hole hopping charge recombination in the CcP:Cc complex. Experimental studies find that when Trp191 is substituted by tyrosine, phenylalanine, or redox-active aniline derivatives bound in the W191G cavity, enzymatic activity and charge recombination rates both decrease. Theoretical analysis of these CcP:Cc complexes finds that the ET kinetics depend strongly on the chemistry of the modified Trp site. The computed electronic couplings in the W191F and W191G species are orders of magnitude smaller than in the native protein, due largely to the absence of a hopping intermediate and the large tunneling distance. Small molecules bound in the W191G cavity are weakly coupled electronically to the Cc heme, and the structural disorder of the guest molecule in the binding pocket may contribute further to the lack of enzymatic activity. The couplings in W191Y are not substantially weakened compared to the native species, but the redox potential difference for tyrosine vs tryptophan oxidation accounts for the slower rate in the Tyr mutant. Thus, theoretical analysis explains why only the native Trp supports rapid hole hopping in the CcP:Cc complex. Favorable free energies and electronic couplings are essential for establishing an efficient hole hopping relay in this protein-protein complex.

The redox potential of a heme cofactor in Nitrosomonas europaea cytochrome c peroxidase: a polarizable QM/MM study.

Redox reactions are crucial to biological processes that protect organisms against oxidative stress. Metalloenzymes, such as peroxidases which reduce excess reactive oxygen species into water, play a key role in detoxification mechanisms. Here we present the results of a polarizable QM/MM study of the reduction potential of the electron transfer heme in the cytochrome c peroxidase of Nitrosomonas europaea. We have found that environment polarization does not substantially affect the computed value of the redox potential. Particular attention has been given to analyzing the role of electrostatic interactions within the protein environment and the solvent on tuning the redox potential of the heme co-factor. We have found that the electrostatic interactions predominantly explain the fluctuations of the vertical ionization/attachment energies of the heme for the sampled configurations, and that the long range electrostatic interactions (up to 40 Å) contribute substantially to the absolute values of the vertical energy gaps.

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids.

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

Extra copy of the mitochondrial cytochrome-c peroxidase gene confers a pyruvate-underproducing characteristic of sake yeast through respiratory metabolism.

The mechanism of pyruvate-underproduction of aneuploid sake yeast was investigated in this study. In our previous report, we revealed that an increase in chromosome XI decreases pyruvate productivity of sake yeast. In this report, we found that increased copy number of CCP1, which is located on chromosome XI and encodes cytochrome-c peroxidase, decreased the pyruvate productivity of sake yeasts. Introducing an extra copy of CCP1 activated respiratory metabolism governed by Hap4 and increased reactive oxygen species. Therefore, it was concluded that increased copy number of CCP1 on chromosome XI activated respiratory metabolism and decreased pyruvate levels in an aneuploid sake yeast. This is the first report that describes a mechanism underlying the improvement of brewery yeast by chromosomal aneuploidy.

Methylglyoxal-Scavenging Enzyme Activities Trigger Erythroascorbate Peroxidase and Cytochrome c Peroxidase in Glutathione-Depleted Candida albicans.

γ-Glutamylcysteine synthetase (Gcs1) and glutathione reductase (Glr1) activity maintains minimal levels of cellular methylglyoxal in Candida albicans. In glutathione-depleted Δgcs1, we previously saw that NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase (Adh1) are the most active methylglyoxal scavengers. With methylglyoxal accumulation, disruptants lacking MGD1 or ADH1 exhibit a poor redox state. However, there is little convincing evidence for a reciprocal relationship between methylglyoxal scavenger genes-disrupted mutants and changes in glutathione-(in)dependent redox regulation. Herein, we attempt to demonstrate a functional role for methylglyoxal scavengers, modeled on a triple disruptant (Δmgd1/Δadh1/Δgcs1), to link between antioxidative enzyme activities and their metabolites in glutathione-depleted conditions. Despite seeing elevated methylglyoxal in all of the disruptants, the result saw a decrease in pyruvate content in Δmgd1/Δadh1/Δgcs1 which was not observed in double gene-disrupted strains such as Δmgd1/Δgcs1 and Δadh1/Δgcs1. Interestingly, Δmgd1/Δadh1/Δgcs1 exhibited a significantly decrease in H2O2 and superoxide which was also unobserved in Δmgd1/Δgcs1 and Δadh1/Δgcs1. The activities of the antioxidative enzymes erythroascorbate peroxidase and cytochrome c peroxidase were noticeably higher in Δmgd1/Δadh1/Δgcs1 than in the other disruptants. Meanwhile, Glr1 activity severely diminished in Δmgd1/Δadh1/Δgcs1. Monitoring complementary gene transcripts between double gene-disrupted Δmgd1/Δgcs1 and Δadh1/Δgcs1 supported the concept of an unbalanced redox state independent of the Glr1 activity for Δmgd1/Δadh1/Δgcs1. Our data demonstrate the reciprocal use of Eapx1 and Ccp1 in the absence of both methylglyoxal scavengers; that being pivotal for viability in non-filamentous budding yeast.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome†c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold.

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

A Stable Ferryl Porphyrin at the Active Site of Y463M BthA.

BthA is a diheme enzyme that is a member of the bacterial cytochrome c peroxidase superfamily, capable of generating a highly unusual Fe(IV)Fe(IV)═O oxidation state, known to be responsible for long-range oxidative chemistry in the enzyme MauG. Here, we show that installing a canonical Met ligand in lieu of the Tyr found at the heme of MauG associated with electron transfer, results in a construct that yields an unusually stable Fe(IV)═O porphyrin at the peroxidatic heme. This state is spontaneously formed at ambient conditions using either molecular O2 or H2O2. The resulting data illustrate how a ferryl iron, with unforeseen stability, may be achieved in biology.

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy.

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.

Structure elaboration of isoniazid: synthesis, in silico molecular docking and antimycobacterial activity of isoniazid-pyrimidine conjugates.

Designing small molecule-based new drug candidates through structure modulation of the existing drugs has drawn considerable attention in view of inevitable emergence of resistance. A new series of isoniazid-pyrimidine conjugates were synthesized in good yields and evaluated for antitubercular activity against the H37Rv strain of Mycobacterium tuberculosis using the microplate Alamar Blue assay. Structure-anti-TB relationship profile revealed that conjugates 8a and 8c bearing a phenyl group at C-6 of pyrimidine scaffold were most active (MIC99 10 µM) and least cytotoxic members of the series. In silico docking of 8a in the active site of bovine lactoperoxidase as well as a cytochrome C peroxidase mutant N184R Y36A revealed favorable interactions similar to the heme enzyme catalase peroxidase (KatG) that activates isoniazid. This investigation suggests a rationale for further work on this promising series of antitubercular agents.

Dual species transcript profiling during the interaction between banana (Musa acuminata) and the fungal pathogen Fusarium oxysporum f. sp. cubense.

BACKGROUND: Banana wilt disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), is one of the most devastating diseases in banana (Musa spp.). Foc is a soil borne pathogen that causes rot of the roots or wilt of leaves by colonizing the xylem vessels. The dual RNA sequencing is used to simultaneously assess the transcriptomes of pathogen and host. This method greatly helps to understand the responses of pathogen and host to each other and discover the potential pathogenic mechanism. RESULTS: Plantlets of two economically important banana cultivars, Foc TR4 less susceptible cultivar NK and susceptible cultivar BX, were used to research the Foc-banana interaction mechanism. Notably, the infected NK had more significantly up-regulated genes on the respiration machinery including TCA cycle, glyoxylate, glycerol, and glycolysis compared to BX at 27 h post inoculation (hpi). In addition, genes involved in plant-pathogen interaction, starch, sucrose, linolenic acid and sphingolipid metabolisms were uniquely more greatly induced in BX than those in NK during the whole infection. Genes related to the biosynthesis and metabolism of SA and JA were greatly induced in the infected NK; while auxin and abscisic acid metabolisms related genes were strongly stimulated in the infected BX at 27 hpi. Furthermore, most of fungal genes were more highly expressed in the roots of BX than in those of NK. The fungal genes related to pathogenicity, pectin and chitin metabolism, reactive oxygen scavenging played the important roles during the infection of Foc. CCP1 (cytochrome c peroxidase 1) was verified to involve in cellulose utilization, oxidative stress response and pathogenicity of fungus. CONCLUSION: The transcriptome indicated that NK had much faster defense response against Foc TR4 than BX and the expression levels of fungal genes were higher in BX than those in NK. The metabolisms of carbon, nitrogen, and signal transduction molecular were differentially involved in pathogen infection in BX and NK. Additionally, the putative virulence associated fungal genes involved in colonization, nutrition acquirement and transport provided more insights into the infection process of Foc TR4 in banana roots.

Reduction of hydrogen peroxide in gram-negative bacteria - bacterial peroxidases.

Bacteria display an array of enzymes to detoxify reactive oxygen species that cause damage to DNA and to other biomolecules leading to cell death. Hydrogen peroxide is one of these species, with endogenous and exogenous sources, such as lactic acid bacteria, oxidative burst of the immune system or chemical reactions at oxic-anoxic interfaces. The enzymes that detoxify hydrogen peroxide will be the focus of this review, with special emphasis on bacterial peroxidases that reduce hydrogen peroxide to water. Bacterial peroxidases are periplasmic cytochromes with either two or three c-type haems, which have been classified as classical and non-classical bacterial peroxidases, respectively. Most of the studies have been focus on the classical bacterial peroxidases, showing the presence of a reductive activation in the presence of calcium ions. Mutagenesis studies have clarified the catalytic mechanism of this enzyme and were used to propose an intramolecular electron transfer pathway, with far less being known about the intermolecular electron transfer that occurs between reduced electron donors and the enzyme. The physiological function of these enzymes was not very clear until it was shown, for the non-classical bacterial peroxidase, that this enzyme is required for the bacteria to use hydrogen peroxide as terminal electron acceptor under anoxic conditions. These non-classical bacterial peroxidases are quinol peroxidases that do not require reductive activation but need calcium ions to attain maximum activity and share similar catalytic intermediates with the classical bacterial peroxidases.

Resonance Raman, Electron Paramagnetic Resonance, and Magnetic Circular Dichroism Spectroscopic Investigation of Diheme Cytochrome c Peroxidases from Nitrosomonas europaea and Shewanella oneidensis.

Cytochrome c peroxidases (bCcPs) are diheme enzymes required for the reduction of H2O2 to water in bacteria. There are two classes of bCcPs: one is active in the diferric form (constitutively active), and the other requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). To improve our understanding of the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( NeCcP) and a reductively activated bCcP from Shewanella oneidensis ( SoCcP) were characterized in both the diferric and semireduced states by electron paramagnetic resonance (EPR), resonance Raman (rRaman), and magnetic circular dichroism (MCD) spectroscopy. In contrast to some previous crystallographic studies, EPR and rRaman spectra do not indicate the presence of significant amounts of a five-coordinate, high-spin ferric heme in NeCcP or SoCcP in either the diferric or semireduced state in solution. This observation points toward a mechanism of activation in which the active site L-heme is not in a static, five-coordinate state but where the activation is more subtle and likely involves formation of a six-coordinate hydroxo complex, which could then react with hydrogen peroxide in an acid-base-type reaction to create Compound 0, the ferric hydroperoxo complex. This mechanism lies in stark contrast to the diheme enzyme MauG that exhibits a static, five-coordinate open heme site at the peroxidatic heme and that forms a more stable FeIV═O intermediate.

LC-MS/MS Proteoform Profiling Exposes Cytochrome c Peroxidase Self-Oxidation in Mitochondria and Functionally Important Hole Hopping from Its Heme.

LC-MS/MS profiling reveals that the proteoforms of cytochrome c peroxidase (Ccp1) isolated from respiring yeast mitochondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccp1 by H2O2 in the absence of its reducing substrate, ferrocytochrome c, gives rise to similar proteoforms, indicating uncoupling of Ccp1 oxidation and reduction in mitochondria. The oxidative modifications found in the Ccp1 proteoforms are consistent with radical transfer (hole hopping) from the heme along several chains of redox-active residues (Trp, Met, Tyr). These modifications delineate likely hole-hopping pathways to novel substrate-binding sites. Moreover, a decrease in recombinant Ccp1 oxidation by H2O2 in vitro in the presence of glutathione supports a protective role for hole hopping to this antioxidant. Isolation and characterization of extramitochondrial Ccp1 proteoforms reveals that hole hopping from the heme in these proteoforms results in selective oxidation of the proximal heme ligand (H175) and heme labilization. Previously, we demonstrated that this labilized heme is recruited for catalase maturation (Kathiresan, M.; Martins, D.; English, A. M. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17468-17473; DOI: 10.1073/pnas.1409692111 ). Following heme release, apoCcp1 exits mitochondria, yielding the extramitochondrial proteoforms that we characterize here. The targeting of Ccp1 for selective H175 oxidation may be linked to the phosphorylation status of Y153 close to the heme since pY153 is abundant in certain proteoforms. In sum, when insufficient electrons from ferrocytochrome c are available to Ccp1 in mitochondria, hole hopping from its heme expands its physiological functions. Specifically, we observe an unprecedented hole-hopping sequence for heme labilization and identify hole-hopping pathways from the heme to novel substrates and to glutathione at Ccp1's surface. Furthermore, our results underscore the power of proteoform profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.

The proportion of Met80-sulfoxide dictates peroxidase activity of human cytochrome c.

The peroxidase activity of cytochrome c is proposed to contribute to apoptosis by peroxidation of cardiolipin in the mitochondrial inner membrane. However, cytochrome c heme is hexa-coordinate with a methionine (Met80) on the distal side, stopping it from acting as an efficient peroxidase. The first naturally occurring variant of cytochrome c discovered, G41S, has higher peroxidase activity than wild-type. To understand the basis for this increase and gain insight into the peroxidase activity of wild-type, we have studied wild-type, G41S and the unnatural variant G41T. Through a combined kinetic and mass spectrometric analysis, we have shown that hydrogen peroxide specifically oxidizes Met80 to the sulfoxide. In the absence of substrate this can be further oxidized to the sulfone, leading to a decrease in peroxidase activity. Peroxidase activity can be correlated with the proportion of sulfoxide present and if fully in that form, all variants have the same activity without a lag phase caused by activation of the protein.

Interaction between Neisseria gonorrhoeae bacterial peroxidase and its electron donor, the lipid-modified azurin.

The Neisseria gonorrhoeae bacterial cytochrome c peroxidase plays a key role in detoxifying the cells from H2 O2 by reducing it to water using the lipid-modified azurin, LAz, a small type 1 copper protein, as electron donor. Here, the interaction between these two proteins was characterized by steady-state kinetics, two-dimensional NMR and molecular docking simulations. LAz is an efficient electron donor capable of activating this enzyme. This electron transfer complex is weak with a hydrophobic character, with LAz binding close to the electron transferring heme of the enzyme. The high catalytic rate (39 ± 0.03 s-1 ) is explained by the LAz pre-orientation, due to a positive dipole moment, and by the fast-dynamic ensemble of orientations, suggested by the small chemical shifts.