Cytochrome C Peroxidase Research Articles

Constraints on the Radical Cation Center of Cytochrome c Peroxidase for Electron Transfer from Cytochrome c.

The tryptophan 191 cation radical of cytochrome c peroxidase (CcP) compound I (Cpd I) mediates long-range electron transfer (ET) to cytochrome c (Cc). Here we test the effects of chemical substitution at position 191. CcP W191Y forms a stable tyrosyl radical upon reaction with peroxide and produces spectral properties similar to those of Cpd I but has low reactivity toward reduced Cc. CcP W191G and W191F variants also have low activity, as do redox ligands that bind within the W191G cavity. Crystal structures of complexes between Cc and CcP W191X (X = Y, F, or G), as well as W191G with four bound ligands reveal similar 1:1 association modes and heme pocket conformations. The ligands display structural disorder in the pocket and do not hydrogen bond to Asp235, as does Trp191. Well-ordered Tyr191 directs its hydroxyl group toward the porphyrin ring, with no basic residue in the range of interaction. CcP W191X (X = Y, F, or G) variants substituted with zinc-porphyrin (ZnP) undergo photoinduced ET with Cc(III). Their slow charge recombination kinetics that result from loss of the radical center allow resolution of difference spectra for the charge-separated state [ZnP(+), Cc(II)]. The change from a phenyl moiety at position 191 in W191F to a water-filled cavity in W191G produces effects on ET rates much weaker than the effects of the change from Trp to Phe. Low net reactivity of W191Y toward Cc(II) derives either from the inability of ZnP(+) or the Fe-CcP ferryl to oxidize Tyr or from the low potential of the resulting neutral Tyr radical.

Cytochrome c peroxidase activity of heme bound amyloid β peptides.

Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP.

Scanning Electrochemical Microscopy of Cytochrome c Peroxidase through the Orientation‐Controlled Immobilisation of Cytochrome c

AbstractImmobilisation of enzymes onto surfaces with defined orientation is of particular importance in order to optimise their activity and capacity to dock electron‐transfer partners. Local characterisation methods are thus crucial to understanding the activity–orientation relationship of immobilised enzymes for long‐term storage and applications. Herein, cytochrome c (cyt c) is chemoselectively immobilised through a cysteine functional group with a maleimide‐functionalised gold surface, thus controlling cyt c orientation. X‐ray photoelectron spectroscopy confirmed successful cyt c immobilisation. Scanning electrochemical microscopy methodologies were developed to quantify the activity of immobilised cyt c and cytochrome c peroxidase (CcP). The results suggest optimal enzymatic activities and docking ability for surface‐tethered cyt c are obtained through the cysteine–maleimide linkage, in comparison to conventional indiscriminate lysine–carboxyl linkages. This study reveals the strong influence of cyt c orientation, as a result of linking strategy, upon the docking ability of redox‐electron partner CcP.

Weak self-association of cytochrome c peroxidase molecules observed by paramagnetic NMR.

There is growing experimental evidence that many proteins exhibit a tendency for (ultra)weak homo- or hetero- oligomerization interactions. With the development of paramagnetic relaxation enhancement NMR spectroscopy it has become possible to characterize weak complexes experimentally and even detect complexes with affinities in the 1–25 mM range. We present evidence for a weak complex between cytochrome c peroxidase (CcP) molecules. In a previous study, we attached nitroxide based spin labels at three positions on CcP with the intent of observing intramolecular PRE effects. However, several intermolecular PRE effects were also observed suggesting a weak self-association between CcP molecules. The CcP–CcP complex was characterized using paramagnetic NMR and protein docking. The interaction occurs between the surface that is also part of the stereo-specific binding site for its physiological partner, cytochrome c (Cc), and several small, positively charged patches on the “back” of CcP. The CcP–CcP complex is not a stereo-specific complex. It is a dynamic ensemble of orientations, characteristic of an encounter state. The contact areas resemble those observed for CcP molecules in crystals. The CcP–CcP complex formation competes with that of the CcP-Cc complex. However, the affinity for Cc is much larger and thus it is expected that, under physiological conditions, auto-inhibition will be limited.Graphical A weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0035-z) contains supplementary material, which is available to authorized users.

Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer

The reaction of peroxides with peroxidases oxidizes the heme iron from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical. X-ray-generated hydrated electrons rapidly reduce Fe(IV), thereby requiring very short exposures using many crystals, and, even then, some reduction cannot be avoided. The new generation of X-ray free electron lasers capable of generating intense X-rays on the tenths of femtosecond time scale enables structure determination with no reduction or X-ray damage. Here, we report the 1.5-Å crystal structure of cytochrome c peroxidase (CCP) compound I (CmpI) using data obtained with the Stanford Linear Coherent Light Source (LCLS). This structure is consistent with previous structures. Of particular importance is the active site water structure that can mediate the proton transfer reactions required for both CmpI formation and reduction of Fe(IV)=O to Fe(III)-OH. The structures indicate that a water molecule is ideally positioned to shuttle protons between an iron-linked oxygen and the active site catalytic His. We therefore have carried out both computational and kinetic studies to probe the reduction of Fe(IV)=O. Kinetic solvent isotope experiments show that the transfer of a single proton is critical in the peroxidase rate-limiting step, which is very likely the proton-coupled reduction of Fe(IV)=O to Fe(III)-OH. We also find that the pKa of the catalytic His substantially increases in CmpI, indicating that this active site His is the source of the proton required in the reduction of Fe(IV)=O to Fe(IV)-OH.

Structure and Function of Transient Encounters of Redox Proteins.

Many biomolecular interactions proceed via lowly populated, transient intermediates. Believed to facilitate formation of a productive complex, these short-lived species are inaccessible to conventional biophysical and structural techniques and, until recently, could only be studied by theoretical simulations. Recent development of experimental approaches sensitive to the presence of minor species--in particular paramagnetic relaxation enhancement (PRE) NMR spectroscopy--has enabled direct visualization and detailed characterization of such lowly populated states. Collectively referred to as an encounter complex, the binding intermediates are particularly important in transient protein interactions, such as those orchestrating signaling cascades or energy-generating electron transfer (ET) chains. Here I discuss encounter complexes of redox proteins mediating biological ET reactions, which are essential for many vital cellular activities including oxidative phosphorylation and photosynthesis. In particular, this Account focuses on the complex of cytochrome c (Cc) and cytochrome c peroxidase (CcP), which is a paradigm of biomolecular ET and an attractive system for studying protein binding and enzymatic catalysis. The Cc-CcP complex formation proceeds via an encounter state, consisting of multiple protein-protein orientations sampled in the search of the dominant, functionally active bound form and exhibiting a broad spatial distribution, in striking agreement with earlier theoretical simulations. At low ionic strength, CcP binds another Cc molecule to form a weak ternary complex, initially inferred from kinetics experiments and postulated to account for the measured ET activity. Despite strenuous efforts, the ternary complex could not be observed directly and remained eagerly sought for the past two decades. Very recently, we have solved its structure in solution and shown that it consists of two binding forms: the dominant, ET-inactive geometry and an ensemble of lowly populated species with short separations between Cc and CcP cofactors, which summarily account for the measured ET rate. Unlike most protein complexes, which require accurate alignment of the binding surfaces in a single, well-defined orientation to carry out their function, redox proteins can form multiple productive complexes. As fast ET will occur any time the redox centers of the binding partners are close enough to ensure efficient electron tunneling across the interface, many protein-protein orientations are expected to be ET active. The present analysis confirms that the low-occupancy states can support the functional ET activity and contribute to the stability of redox protein complexes. As illustrated here, boundaries between the dominant and the encounter forms become blurred for many dynamic ET systems, which are more aptly described by ensembles of functionally and structurally heterogeneous bound forms.

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.

Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins

Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

Binding of Yeast Cytochrome c to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome c Peroxidase: Isothermal Titration Calorimetry.

Previously, we constructed, expressed, and purified 46 charge-reversal mutants of yeast cytochrome c peroxidase (CcP) and determined their electronic absorption spectra, their reaction with H2O2, and their steady-state catalytic properties [ Pearl , N. M. et al. (2008) Biochemistry 47 , 2766 - 2775 ]. Forty-four of the mutants involve the conversion of either an aspartate or glutamate residue to a lysine residue, while two are positive-to-negative mutations, R31E and K149D. In this paper, we report on a calorimetric study of the interaction of each charge-reversal mutant (excluding the internal mutants D76K and D235K) with recombinant yeast iso-1 ferricytochrome c(C102T) (yCc) under conditions where only one-to-one yCc/CcP complex formation is observed. Thirteen of the 44 surface-site charge-reversal mutants decrease the binding affinity for yCc by a factor of 2 or more. Eight of the 13 mutations (E32K, D33K, D34K, E35K, E118K, E201K, E290K, E291K) occur within, or on the immediate periphery, of the crystallographically defined yCc binding site [ Pelletier , H. and Kraut , J. (1992) Science 258 , 1748 - 1755 ], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the "back-side" of CcP. The current study is consistent with a model for yCc binding to CcP in which yCc binds predominantly near the region defined by crystallographic structure of the 1:1 yCc-CcP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex.

Enzymatic Mechanism of Leishmania major Peroxidase and the Critical Role of Specific Ionic Interactions.

Leishmania major peroxidase (LmP) is very similar to the well-known yeast cytochrome c peroxidase (CcP). Both enzymes catalyze the peroxidation of cytochrome c. Like CcP, LmP reacts with H2O2 to form Compound I, which consists of a ferryl heme and a Trp radical, Fe(IV)═O;Trp(•+). Cytochrome c (Cytc) reduces the Trp radical to give Compound II, Fe(IV)═O;Trp, which is followed by an intramolecular electron transfer to give Fe(III)-OH;Trp(•+), and in the last step, Cytc reduces the Trp radical. In this study, we have used steady-state and single-turnover kinetics to improve our understanding of the overall mechanism of LmP catalysis. While the activity of CcP greatly increases with ionic strength, the kcat for LmP remains relatively constant at all ionic strengths tested. Therefore, unlike CcP, where dissociation of oxidized Cytc is limiting at low ionic strengths, association/dissociation reactions are not limiting at any ionic strength in LmP. We conclude that in LmP, the intramolecular electron transfer reaction, Fe(IV)═O;Trp to Fe(III)-OH;Trp(•+), is limiting at all ionic strengths. Unlike CcP, LmP depends on key intermolecular ion pairs to form the electron transfer competent complex. Mutating these sites causes the initial rate of association to decrease by 2 orders of magnitude and a substantial decrease in kcat. The drop in kcat is due to a switch in the rate-limiting step of the mutants from intramolecular electron transfer to the rate of association in forming the LmP-LmCytc complex. These studies show that while LmP and CcP form very similar complexes and exhibit similar activities, they substantially differ in how their activity changes as a function of ionic strength. This difference is primarily due to the heavy reliance of LmP on highly specific intermolecular ion pairs, while CcP relies mainly on nonpolar interactions.

Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L)

Imidazole, 1-methylimidazole and 4-nitroimidazole bind to yeast cytochrome c peroxidase (yCcP) with apparent equilibrium dissociation constants (KDapp) of 3.3±0.4, 0.85±0.11, and ~0.2M, respectively, at pH7. This is the weakest imidazole binding to a heme protein reported to date and it is about 120 times weaker than imidazole binding to metmyoglobin. Spectroscopic changes associated with imidazole and 1-methylimidazole binding to yCcP suggest partial ionization of bound imidazole to imidazolate. The pKa for ionization of bound imidazole is estimated to be 7.4±0.2, about 7 units lower than that of free imidazole and about 3 units lower than imidazole bound to metmyoglobin. Equilibrium binding of imidazole to CcP(H52L) is biphasic with low- and high-affinity phases having KDapp values of 9.5±4.5 and 0.13±0.04M, respectively. CcP(H52L) binding of 1-methylimidazole is monophasic with an affinity similar to those of yCcP and rCcP. Binding of 1-methylimidazole to rCcP is associated with two kinetic phases, the initial binding complete within 10s, followed by a process that is consistent with 1-methylimidazole binding to a cavity created by movement of Trp-191 from the interior of the protein to the surface. Both the equilibrium binding and kinetics of 1-methylimidazole binding to yCcP are pH dependent. yCcP has a four-fold increase in 1-methylimidazole binding affinity on decreasing the pH from 7.5 to 4.0, an observation that is unique among the many studies on binding of imidazole and imidazole derivatives to heme proteins.

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants

Imidazole binding to three apolar distal heme pocket mutants of yeast cytochrome c peroxidase (CcP) has been investigated between pH4 and 8. The three CcP variants have Arg-48, Trp-51, and His-52 mutated to either all alanine, CcP(triAla), all valine, CcP(triVal), or all leucine residues, CcP(triLeu). The imidazole binding curves for all three mutants are biphasic indicating that each of the mutants exists in at least two conformational states with different affinities for imidazole. At pH7, the high-affinity conformations of the three CcP mutants bind imidazole between 3.8 and 4.7 orders of magnitude stronger than that of wild-type CcP while the low-affinity conformations have binding affinities about 2.5 orders of magnitude larger than wild-type CcP. Imidazole binding to the three CcP mutants is pH dependent with the strongest binding observed at high pH. Apparent pKa values for the transition in binding vary between 5.6 and 7.5 for the high-affinity conformations and between 6.2 and 6.8 for the low-affinity conformations of the CcP triple mutants. The kinetics of imidazole binding are also biphasic. The fast phase of imidazole binding to CcP(triAla) and CcP(triLeu) is linearly dependent on the imidazole concentration while the slow phase is independent of imidazole concentration. Both phases of imidazole binding to CcP(triVal) have a hyperbolic dependence on the imidazole concentration. The apparent association rate constants vary between 30 and 170M−1s−1 while the apparent dissociation rate constants vary between 0.05 and 0.43s−1. The CcP triple mutants have higher binding affinities for 1-methylimidazole and 4-nitroimidazole than does wild-type CcP.

Control of cyclic photoinitiated electron transfer between cytochrome c peroxidase (W191F) and cytochrome c by formation of dynamic binary and ternary complexes.

Extensive studies of the physiological protein-protein electron-transfer (ET) complex between yeast cytochrome c peroxidase (CcP) and cytochrome c (Cc) have left unresolved questions about how formation and dissociation of binary and ternary complexes influence ET. We probe this issue through a study of the photocycle of ET between Zn-protoporphyrin IX-substituted CcP(W191F) (ZnPCcP) and Cc. Photoexcitation of ZnPCcP in complex with Fe(3+)Cc initiates the photocycle: charge-separation ET, [(3)ZnPCcP, Fe(3+)Cc] → [ZnP(+)CcP, Fe(2+)Cc], followed by charge recombination, [ZnP(+)CcP, Fe(2+)Cc] → [ZnPCcP, Fe(3+)Cc]. The W191F mutation eliminates fast hole hopping through W191, enhancing accumulation of the charge-separated intermediate and extending the time scale for binding and dissociation of the charge-separated complex. Both triplet quenching and the charge-separated intermediate were monitored during titrations of ZnPCcP with Fe(3+)Cc, Fe(2+)Cc, and redox-inert CuCc. The results require a photocycle that includes dissociation and/or recombination of the charge-separated binary complex and a charge-separated ternary complex, [ZnP(+)CcP, Fe(2+)Cc, Fe(3+)Cc]. The expanded kinetic scheme formalizes earlier proposals of "substrate-assisted product dissociation" within the photocycle. The measurements yield the thermodynamic affinity constants for binding the first and second Cc: KI = 10(-7) M(-1), and KII = 10(-4) M(-1). However, two-site analysis of the thermodynamics of formation of the ternary complex reveals that Cc binds at the weaker-binding site with much greater affinity than previously recognized and places upper bounds on the contributions of repulsion between the two Cc's of the ternary complex. In conjunction with recent nuclear magnetic resonance studies, the analysis further suggests a dynamic view of the ternary complex, wherein neither Cc necessarily faithfully adopts the crystal-structure configuration because of Cc-Cc repulsion.

In silico spectroscopy of tryptophan and tyrosine radicals involved in the long-range electron transfer of cytochrome c peroxidase.

Cytochrome c peroxidase (CcP) is a heme-containing enzyme that catalyzes the oxidation of the ferrocytochrome c to ferricytochrome c with concomitant reduction of H2O2 to H2O. Its catalytic cycle involves the formation of a double oxidized species (compound I) consisting of an oxoferryl center (Fe(IV)═O) and an amino acid radical (R(•)). Here we use a quantum-mechanics/molecular-mechanics (QM/MM) computational protocol based on density functional theory (DFT) and multiconfigurational perturbation theory (CASPT2) methods to reproduce specific features of compound I EPR and UV-vis spectra. The results show that the employed QM/MM models can correctly predict the magnetic, electronic and vibrational properties of the observed amino acid radicals of compound I. Furthermore, we have been able to confirm that the principal radical species of compound I is a tryptophan cationic radical located on residue 191 (Trp191(•+)) and that three tyrosine residues (Tyr203, Tyr236, and Tyr251), located along two possible ET pathways involving Trp191(•+), are possible candidates to host the secondary radical species.

Cofactor-dependent structural and binding properties of yeast cytochrome C peroxidase.

Yeast cytochrome c peroxidase (CcP) is a heme enzyme that reduces hydroperoxides using the electrons provided by its physiological partner cytochrome c (Cc). Contributing to the resistance against the oxidative stress associated with the aerobic metabolism, the Cc-CcP complex has been widely studied and became a paradigm for biological electron transfer. The heme-free, enzymatically inactive apo CcP is the natural precursor of the mature, cofactor-bound holo protein. Despite its physiological relevance, apo CcP is not well characterized, and at present, little is known about its structure or the interaction with Cc. Using a range of biophysical techniques, here we show that, while holo CcP binds Cc with micromolar affinity, the interaction between apo CcP and Cc is completely abolished. Characterized by small-angle X-ray scattering, solution nuclear magnetic resonance spectroscopy, and equilibrium unfolding experiments, apo and holo CcP exhibit very similar structural, hydrodynamic, and thermodynamic properties. However, detailed analysis reveals that apo CcP is more expanded in solution, displays a number of characteristics associated with a molten globule state, and, unlike the holo protein, does not form an unfolding intermediate during thermal and chemical denaturation. Overall, our data suggest that the Cc binding site present in the holo protein is disrupted in the apo form, explaining the inability of the latter to interact with Cc. We argue that the observed difference in Cc binding is physiologically relevant and suggest why abolishing the apo CcP-Cc interaction is beneficial to the organism.

Identifying the elusive sites of tyrosyl radicals in cytochrome c peroxidase: implications for oxidation of substrates bound at a site remote from the heme.

The location of the Trp radical and the catalytic function of the [Fe(IV)=O Trp191•+] intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr71• and Tyr236• is independent of the [Fe(IV)=O Trp191•+] intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Å from the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632–1639]. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr71• as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking [Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248–255] and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole [Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665–11674]. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction [Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557–8563]. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.

The cytochrome c peroxidase and cytochrome c encounter complex: the other side of the story.

Formation of an encounter complex is important for efficient protein complex formation. The encounter state consists of an ensemble of orientations of two proteins in the complex. Experimental description of such ensembles inherently suffers from insufficient data availability. We have measured paramagnetic relaxation enhancements (PRE) on cytochrome c peroxidase (CcP) caused by its partner cytochrome c (Cc) carrying a spin label. The data complement earlier PRE data of spin labelled CcP, identifying several new interactions. This work demonstrates the need of obtaining as many independent data sets as possible to achieve the most accurate description of an encounter complex.

A novel colorimetric method for the detection ofEscherichia coliusing cytochrome c peroxidase-encoding bacteriophage

A new rapid and simple method was developed for the detection of Escherichia coli by constructing a recombinant T4 phage carrying the cytochrome c peroxidase gene derived from Saccharomyces cerevisiae (T4ccp) using which, the colorimetric detection of E. coli K12 was examined. The oxidation activity toward the chromogenic substrate cytochrome c was demonstrated by the cytochrome c peroxidase (CCP) produced from the T4ccp genome. The color change caused by the oxidation of the substrate could be visually perceived. The possibility of interference in the detection by the coexistence of other bacteria was assessed using Pseudomonas aeruginosa as a nontarget bacterium, and it was confirmed that the coexistence of P. aeruginosa caused no interference in the detection of E. coli K12.

Artesunate Effect on Schistosome Thioredoxin Glutathione Reductase and Cytochrome c Peroxidase as New Molecular Targets in Schistosoma mansoni-infected Mice

ObjectiveTo investigate the possible effect of artesunate (ART) on schistosome thioredoxin glutathione reductase (TGR) and cytochrome c peroxidase (CcP) in Schistosoma mansoni-infected mice. MethodsA total of 200 laboratory bred male Swiss albino mice were divided into 4 groups (50 mice in each group). Group I: infected untreated group (Control group) received a vehicle of 1% sodium carbonyl methylcellulose (CMC-Na); Group II: infected then treated with artesunate; Group III: infected then treated with praziquantel, and group IV: infected then treated with artesunate then praziquantel. Adult S. mansoni worms were collected by Animal Perfusion Method, tissue egg counted, TGR, and CcP mRNA Expression were estimated of in S. mansoni adult worms by semi-quantitative rt-PCR. ResultsSemi-quantitative rt-PCR values revealed that treatment with artesunate caused significant decrease in expression of schistosome TGR and CcP in comparison to the untreated group. In contrast, the treatment with praziquantel did not cause significant change in expression of these genes. The results showed more reduction in total worm and female worm count in combined ART-PZQ treated group than in monotherapy treated groups by either ART or PZQ. Moreover, complete disappearance (100%) of tissue eggs was recorded in ART-PZQ treated group with a respective reduction rate of 95.9% and 68.4% in ART- and PZQ-treated groups. ConclusionThe current study elucidated for the first time that anti-schistosomal mechanisms of artesunate is mediated via reduction in expression of schistosome TGR and CcP. Linking these findings, addition of artesunate to praziquantel could achieve complete cure outcome in treatment of schistosomiasis.

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16M−1s−1 and 0.34±0.15s−1, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61min−1 at pH 6.0.