Cytochrome C Peroxidase Research Articles

Enhanced oxidation of aniline derivatives by two mutants of cytochrome c peroxidase at tryptophan 51.

Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze the enhanced oxidation of a number of substituted anilines. The reaction of CCP compound ES with mesidine is biphasic, while similar reactions using compound II give monophasic kinetics. These data, in addition to the ratio of the Fe4+ = O and free-radical species observed during steady-state turnover, indicate that reduction of the Trp-191 free radical of compound ES is more rapid than the reduction of the Fe4+ = O species. Transient kinetics were examined for the oxidation of eight mono-substituted anilines by CCP, W51F, and W51A. Each of the aniline derivatives were oxidized by the mutants at rates that exceeded that of the wild-type enzyme, and the rate constant for m-chloroaniline was 400-fold faster for W51F than for wild-type CCP. Variations in the rate constants for the different substrates follow a linear free-energy relationship using the Hammet substituent effect parameter sigma +, implicating electron transfer from the aniline ring in the transition state. For aniline oxidation, the free energy of activation is 3 kcal/mol lower for the mutants than for wild-type CCP, and this is due primarily to an increase in the activation entropy. These results indicate that the enhanced kinetics of W51F and W51A result from a generalized increase in enzyme reactivity characterized by an exo-entropic transition state such as dissociation of bound H2O from the Fe4+ = O center.

What do we know about cytochrome c peroxidase?

What do we know about cytochrome c peroxidase?

Quenching of intrinsic fluorescence of yeast cytochrome c peroxidase by covalently- and noncovalently-bound quenchers.

The intrinsic steady-state fluorescence of the heme enzyme cytochrome c peroxidase (CCP) has been characterized as a probe of its structure in solution. The fluorescence is dominated by tryptophan emission, which has a quantum yield of 7% relative to the tryptophan standard N-acetyltryptophanamide, and an emission maximum at 324 nm indicative of a relatively hydrophobic environment for the fluorescent residues. These fluorescence properties are consistent with the known structure of CCP; six of the seven tryptophan residues are well within quenching distance for efficient Förster energy transfer to the heme, so that the intrinsic fluorescence arises largely from Trp101 which is approximately 26 A from the heme and partially buried. Quenching studies using Cs+, I-, and acrylamide are also consistent with this picture, since the charged species are poor quenchers, but acrylamide, which can penetrate the protein matrix, is a more effective quencher. The intrinsic fluorescence of two CCP derivatives with the quencher pentaammineruthenium(III) covalently attached to His6 and His60 has also been characterized. The His60 derivative, shown by X-ray analysis to be essentially structurally identical to native CCP, is 17% less fluorescent than native CCP, consistent with the quenching expected from distance calculations and the assignment of Trp101 as the major fluorescent center. The observed quenching of 38% in the second derivative is close to that predicted for ruthenation of His6 assuming that Trp101 is the major fluorophore. The fluorescence of compound I of CCP is also reported. This species, which has a ferryl (FeIV = O) heme and a protein radical purportedly on Trp191, exhibits 9% higher fluorescence than native CCP.(ABSTRACT TRUNCATED AT 250 WORDS)

Lignin-degrading peroxidases of Phanerochaete chrysosporium

Lignin and manganese peroxidases are secreted by the basidiomycete Phanerochaete chrysosporium during secondary metabolism. These enzymes play major roles in lignin degradation. The active site amino acid sequence of these lignin-degrading peroxidases is similar to that of horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP). The mechanism by which they oxidize substrates also appears to be the similar. pH has a similar effect on lignin peroxidase compound I formation as on HRP or CcP; however, the p K a controlling compound I formation for lignin peroxidase appears to be much lower. Lignin-degrading peroxidases are able to catalyze the oxidation of substrates with high redox potential. This unique ability is consistent with a heme active site of low electron density, which is indicated by high redox potential.

The electrocatalytic enzyme function of adsorbed cytochrome c peroxidase on pyrolytic graphite

Cytochrome c peroxidase (CCP) is a well characterized redox enzyme which can serve as a model for understanding the electrocatalytic function of adsorbed enzymes. Rotating disk electrode (RDE) voltammetry and cyclic voltammetry were used to show that CCP adsorbed on edge-oriented pyrolytic graphite reduces H 2O 2 electrocatalytically in a manner similar but not identical to solution CCP. Bulk electrolysis and Koutecky—Levich analysis of RDE data proved that the observed amperometric signal corresponds to the unmediated 2e − reduction of H 2O 2 to water. The pH dependence of the enzyme—substrate kinetics of adsorbed CCP were similar to those for solution CCP at pH ≤ 6 but showed some differences at pH > 6. RDE studies in solutions of varying ionic strength show that CCP remains strongly adsorbed to pyrolytic graphite even at high ionic strength. Preliminary cyclic voltammetry experiments revealed that adsorbed CCP is reversibly inhibited by inhibitors of solution CCP.

Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH.

Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70. This behavior is similar to that exhibited by the ferrous form of the enzyme. However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP. Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70. In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min). F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction. Horseradish peroxidase, however, does not. The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket. Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme. We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.

Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.

Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175-->Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191-->Phe mutant exhibits only < 0.05% activity. However, the double mutant, His175-->Gln/Phe191-->Phe, exhibits 20% wild type activity. Since it is known that the Trp191-->Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.

Monooxygenase activity of cytochrome c peroxidase.

Recombinant cytochrome c peroxidase (CcP) and a W51A mutant of CcP, in contrast to other classical peroxidases, react with phenylhydrazine to give sigma-bonded phenyl-iron complexes. The conclusion that the heme iron is accessible to substrates is supported by the observation that CcP and W51A CcP oxidize thioanisole to the racemic sulfoxide with quantitative incorporation of oxygen from H2O2. Definitive evidence for an open active site is provided by stereoselective epoxidation by both enzymes of styrene, cis-beta-methylstyrene, and trans-beta-methylstyrene. trans-beta-methylstyrene yields exclusively the trans-epoxide, but styrene yields the epoxide and phenylacetaldehyde, and cis-beta-methylstyrene yields both the cis- and trans-epoxides and 1-phenyl-2-propanone. The sulfoxide, stereoretentive epoxides, and 1-phenyl-2-propanone are formed by ferryl oxygen transfer mechanisms because their oxygen atom derives from H2O2. In contrast, the oxygen in the trans-epoxide from the cis-olefin derives primarily from molecular oxygen and is probably introduced by a protein cooxidation mechanism. cis-[1,2-2H]-1-Phenyl-1-propene is oxidized to [1,1-2H]-1-phenyl-2-propanone without a detectable isotope effect on the epoxide:ketone product ratio. The phenyl-iron complex is not formed and substrate oxidation is not observed when the prosthetic group is replaced by delta-meso-ethylheme. CcP thus has a sufficiently open active site to form a phenyl-iron complex, to oxidize thioanisole to the sulfoxide, and to epoxidize styrene and beta-methylstyrene. The results indicate that a ferryl (Fe(IV) = O)/protein radical pair can be coupled to achieve two-electron oxidations. The unique ability of CcP to catalyze monooxygenation reactions does not conflict with its peroxidase function because cytochrome c is oxidized at a distinct surface site (DePillis, G. D., Sishta, B. P., Mauk, A. G., and Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19334-19341).

Comparison of computational models for simulating heme proteins: A study of cytochrome C peroxidase

We have investigated four computational models of the resting state and HOOH-bound complex of a heme protein, cytochrome c peroxidase (CCP), to determine the most efficient methodology that yields reliable results. Previous studies have focused on the overall dynamic structure of proteins and not explicitly on the motion of small molecules and their effect on nearby residues. In this study, the effects of dielectric, boundary constraints, explicit inclusion of solvent, and charges on the heme unit have specifically been examined. For the case studied here, in which interactions with water are very important, it was found that, contrary to commonly accepted procedures, a distance-dependent dielectric (e = 4R) with a constrained protein and no solvent resulted in unacceptably large deviations from more accurate models. In this case, even a buried Trp residue hydrogen bonded to the water network showed large deviations due to the exaggerated motions of the waters. However, use of a constant dielectric (e = 1) with the same constraints resulted in a computationally efficient model that yielded properties very similar to the more accurate models. © 1992 John Wiley & Sons, Inc.

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH.

The ferrous form of native cytochrome c peroxidase (CCP) is known to undergo a reversible transition when titrated over the pH range of 7.00-9.70. This transition produces a conversion from a pentacoordinate high-spin to a hexacoordinate low-spin heme active site and is clearly apparent in the heme optical absorption spectra. Here, we report the characterization of this transition and its effect upon the local heme environment using various optical spectroscopies. The formation of hexacoordinate low-spin heme is interpreted to involve the binding of His-52 at the distal site after the perturbation of the extensive H-bonded network within and around the heme pocket of CCP(II) at alkaline pH. Interestingly, CD investigations of CCP(II) in the far-UV and Soret regions indicate the dissappearance of a single high-spin species and the existence of at least two low-spin species of CCP(II) as the pH is raised above 7.90. Furthermore, transient resonance Raman experiments demonstrate that the hexacoordinate low-spin species can be photolyzed within 10-ns laser pulses, producing a species similar to the low-pH (high-spin) form of CCP(II) at alkaline pH. However, the extent of photolysis is quite pH dependent, with a maximum photodissociation yield at pH = 8.50.

Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.

Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.

Characterization of catalase-negative mutants of methylotrophic yeastHansenula polymorpha

Three recently isolated catalase-negative mutants of Hansenula polymorpha lost the ability to grow on methanol but grew in media containing glucose, ethanol or glycerol. Their incubation in a medium with methanol resulted in an accumulation of hydrogen peroxide and cell death. During growth of a catalase-negative mutant in chemostat on a mixture of methanol and glucose, neither H2O2 accumulation nor cell death were observed up to the molar ratio of 10:1 of the two substrates. Cytochrome-c peroxidase and NADH-peroxidase activities were detected in the cells. In methylotrophic yeasts, catalase seems to be an enzyme characteristic of the metabolism of methanol but not needed for the metabolism of multicarbon substrates. The hydrogen peroxide produced during growth of the mutants on mixed substrates is detoxified by cytochrome-c peroxidase and other peroxidases.

Compound I radical in site-directed mutants of cytochrome c peroxidase as probed by electron paramagnetic resonance and electron-nuclear double resonance

The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrogen peroxide as an electron acceptor for mitochondrial respiration in the yeast Hansenula polymorpha.

Chemostat cultures of a catalase-negative mutant of Hansenula polymorpha CBS 4732 were able to decompose hydrogen peroxide at a high rate. This was apparent from experiments in which the yeast was grown under carbon limitation in chemostat culture on mixtures of glucose and H2O2. The enzyme responsible for H2O2 degradation is probably the mitochondrial enzyme cytochrome c peroxidase (CCP), which was present at very high activities. This enzyme was partially purified and shown to be specific for reduced cytochrome c as an electron donor; no reaction was observed with NAD(P)H. Thus, reducing equivalents for H2O2 degradation by CCP must be provided by the respiratory chain. That H2O2 can act as an electron acceptor for reducing equivalents could be confirmed with experiments in which cells were incubated with ethanol and H2O2 in the absence of oxygen. This resulted in oxidation of ethanol to equimolar amounts of acetate. Energetic aspects of mitochondrial H2O2 decomposition via CCP and the physiological function of CCP in yeasts are discussed.

Purification of cytochrome c peroxidase for monitoring H 2O 2 production

One of the most precise methods of determining hydrogen peroxide (H 2O 2) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H 2O 2 and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H 2O 2 formation, thus decreasing the sensitivity of this method.

Electron Transfer within the Cytochromec-CytochromecPeroxidase Complex: Dependence of the Transient-State and Steady-State Kinetics on Ionic Strength

Abstract Steady-State and transient-state kinetic studies of the cytochrome c peroxidase (CcP) catalyzed oxidation of ferrocytochrome c by hydrogen peroxide at pH 7.5 as a function on ionic strength have been carried out using both horse heart and yeast iso-1 cytochrome c. The ionic strength affects the apparent interaction between cytochrome c and the oxidized intermediates of CcP while the intracomplex electron transfer rates are essentially independent of ionic strength. The maximum enzyme turnover rates for the oxidation of yeast iso-1 and horse heart ferrocytochrome c are 780 ± 130 s−1 and 280 ± 40 s−1, respectively, independent of ionic strength at pH 7.5. The intramolecular electron transfer rates from bound ferrocytochrome c to the heme site in CcP compound I is about 1900 s−1 and 450 s−1 for yeast iso-1 and horse heart cytochrome c, respectively.

Reorganization Energies for Protein-to-Protein Electron Transfer and Interfacial Dynamics: “Playing Fast and Loose”

Abstract Recent work on electron transfer in protein-protein complexes is summarized. Detailed measurements of binding equilibria and binding dynamics have been obtained for several systems: cytochrome c:cytochrome c peroxidase (CCP), cytochrome c:cytochrome b5, and hemoglobin:hemoglobin reductase. For the cytc:ccp system binding and subsequent electron transfer have ben probed using site-directed mutagenesis. These results suggest that binding and recognition involve several, overlapping binding sites. Furthermore, sites exist for binding cytc(II) vs. cytc(III). Thus, electron transfer must involve diffusion along the protein surface which connects the oxidized and reduced sites. Those single site mutants which bind less strongly have a lower diffusion barrier and thus can undergo more rapid electron transfer. This coupling of interfacial motion to reaction may offer a natural explanation for the large reorganization energies observed in biological systems.

Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235 .fwdarw. Asn-235 mutant

Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein [CcP(MI)] and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined. At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase. In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation. Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175. Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP. Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site. All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins. Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI). It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175).

Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase

Resonance Raman spectra are reported for single crystals of cytochrome c peroxidase (CCP) mutants, taken by using a microscope equipped with a variable-temperature stage. The spectra are similar to those observed for the mutant proteins in solution, but there are detectable differences having to do with the coordination and spin state of the heme. The Asn-235 mutant contains a mixture of six-coordinate high- and low-spin states with a detectably higher fraction of the former than in solution. Upon cooling even to 223 K, the heme is converted mostly to the low-spin form. The Phe-191 mutant likewise shows a high/low-spin six-coordinate mixture, together with a preponderant population of five-coordinate heme. Upon cooling, the high-spin six-coordinate population converts immediately to the low-spin form, while the five-coordinate population does so more slowly. This behavior is intermediate between that of native CCP and the Asn-235 mutant, consistent with an ancillary role for the normal Trp-191-Asp-235 H-bond in the proximal anchoring of the heme Fe. The Phe-51 mutant shows a dominant high-spin five-coordinate heme population in the single crystal, whereas in solution the six-coordinate form is dominant. This difference is mimicked by adding 2-methyl-2,4-pentanediol (MPD) to the solution and is attributed to the dehydrating effect of MPD, which is present during crystallization. Upon lowering the temperature, the five-coordinate heme converts partially to a six-coordinate high-spin form.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of Hydrogen Peroxide byPichia pastoris

Hydrogen peroxide in methylotrophic yeasts can be metabolized in at least two distinct ways. Addition of exogenous hydrogen peroxide removes the dependance of catalase on endogenously-produced hydrogen peroxide resulting enhanced rates of alcohol oxidation. Exogenous hydrogen peroxide is also efficiently degraded by cytochrome c peroxidase (CCP), a competitive reaction which does not result in enhanced alcohol oxidation. To overcome the influence of cytochrome c peroxidase, artificial peroxisomes were prepared by coimmobilization of alcohol oxidase and catalase. These artificial peroxisomes mimic the peroxide-induced rate enhancement observed with whole cells.