Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.

Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.     

A kinetic study of the reaction between cytochrome c peroxidase and hydrogen peroxide. Dependence on pH and ionic strength.

The rate of the reaction between cytochrome c peroxidase and hydrogen peroxide was investigated using the stopped-flow technique. The apparent bimolecular rate constant was determined between pH 3.3 and pH 11 as a function of ionic strength. The pH dependence of the apparent bimolecular rate constant can be explained by assuming that two ionizable groups on the enzyme strongly influence the rate of the reaction. At 0.1 M ionic strength, a group with a pKa of 5.5 must be unprotonated and a group with a pKa of 9.8 must be protonated for the enzyme to react rapidly with hydrogen peroxide. The apparent acid dissociation constants depend upon the ionic strength. The true bimolecular rate constant has a value of (4.5 +/- 0.3) X 10(7) M-1 sec-1 and is independent of ionic strength.     

The oxidation of cytochrome c peroxidase by hydrogen peroxide. Characterization of products.

H2O2 reacts with cytochrome c peroxidase in a variety of ways. The initial reaction produces cytochrome c peroxidase Compound I. If more than a 10-fold excess of H2O2 is added to the enzyme, a portion of the H2O2 will react with Compound I to produce molecular oxygen. The remainder oxidizes the heme group and various amino acid residues in the protein. If less than a 10-fold excess of H2O2 is added to the enzyme, essentially all the H2O2 is utilized by oxidation of amino acid residues in the protein. The oxidation of the amino acid residues by H2O2 substantially modifies the reactivity of cytochrome c peroxidase. The modification of reactivity could be the direct result of amino acid oxidation or an indirect result caused by a perturbation of the protein structure at the active site. The products oxidized at pH 8 lose their ability to react with H2O2. The products oxidized at pH4 react with H2O2 but their reactivity toward Fe(CN)4-6 is substantially reduced.     

Mechanism of cytochrome c peroxidase. O-benzoylhydroxylamine as an analog of hydrogen peroxide.

A number of reagents, some of which are electronic analogs of hydrogen peroxide, will replace it in the reactions of cytochrome c peroxidase. These compounds include N-bromosuccinimide, sodium hypochlorite, and the novel oxidizing agent O-benzoylhydroxylamine. If fragments of the oxidant played a functional role in the structure of the oxidized form of the enzyme, it would be expected that the product formed from O-benzoylhydroxylamine would differ from that formed from hydrogen peroxide. The products formed on reaction of the two oxidizing agents with cytochrome c peroxidase are indistinguishable. This results carries implications for the structure of the so-called ES compound. The extension in the range of specific substrates for cytochrome c peroxidase allows identification of the structure which compounds must possess to be oxidizing substrates for the enzyme. A mechanism for the first step of the reaction is suggested. O-Benzoylhydroxylamine is also a reducing agent, and its reaction with the enzyme is analogous to that of hydrogen peroxide with catalase. The final product of the reaction is the inert nitric oxide complex of ferrous cytochrome c peroxidase.     

Structural transitions of carboxymethylated cytochrome c: calorimetric and circular dichroic studies

Circular dichroism and differential scanning calorimetry studies on the unfolding-refolding process of native and carboxymethylated cytochrome c, induced either by temperature or chemical agents, have been performed. The results have shown that the modified protein has a decreased conformational stability with respect to the native state, in agreement with a structure less compact, but still highly folded, which behaves as a thermodynamically stable "intermediate" between native and fully unfolded cytochrome c.     

The cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide. Steady state kinetic mechanism

Initial velocities for the cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide have been measured as functions of both the ferrocytochrome c (0.27-104 microM) and hydrogen peroxide (0.25-200 microM) concentrations at 25 degrees C, 0.01 M ionic strength, and pH 7 in a cacodylate/KNO3 buffer system Eadie-Hofstee plots of the initial velocity as a function of ferrocytochrome c concentration at constant hydrogen peroxide are nonlinear. A mechanism is proposed which includes random addition of the two substrates to the enzyme and a single catalytically active cytochrome c binding site. The mechanism is consistent with prior studies on cytochrome c peroxidase and fits the steady state kinetic data well.     

Complex-formation between cytochrome c and cytochrome c peroxidase. Kinetic studies

1. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are described. Kinetic differences between the older and more recent preparations of the enzyme most probably arise from differences in intrinsic turnover rates. 2. The time-courses of cytochrome c peroxidation by the enzyme follow essentially first-order kinetics in phosphate buffer. Deviations from first-order kinetics occur in acetate buffer, and are due to a higher enzymic turnover rate in this medium accompanied by a greater tendency to autocatalytic peroxidation of cytochrome c. 3. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are interpreted in terms of a mechanism postulating formation of reversible complexes between the peroxidase and both reduced and oxidized cytochrome c. Formation of these complexes is inhibited at high ionic strengths and by polycations. 4. Oxidized cytochrome c can act as a competitive inhibitor of ferrocytochrome c peroxidation by peroxidase. The K(i) for ferricytochrome c is approximately equal to the K(m) for ferrocytochrome c and thus probably accounts for the observed apparent first-order kinetics even at saturating concentrations of ferrocytochrome c. 5. The results are discussed in terms of a possible analogy between the oxidations of cytochrome c catalysed by yeast peroxidase and by mammalian cytochrome oxidase.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.     

Ascorbate peroxidase activity of cytochrome c

Abstract

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10–60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

Steady-state kinetics of yeast cytochrome c peroxidase catalyzed oxidation of inorganic reductants by hydrogen peroxide

Rates of yeast cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) catalyzed oxidation of bis(tripyridine)cobalt(II) ion, penta(amine)pyridineruthenium(II) ion and ferrocyanide ion by hydrogen peroxide have been found to obey the empirical equation: (formula; see text) in the pH range 5 to 8, and at saturating H2O2 concentrations. [( S] and [CcP] are the concentrations of the reductant and the enzyme, respectively.) Values of k2 were found to be independent of the reductant. The term k0[S] is only significant with the cobalt and ruthenium complexes at high pH. The mechanism proposed to account for this rate equation differs significantly from previous mechanistic proposals. In particular, the rate data require the assignment of the rate-limiting step at high substrate concentrations to a slow electron-transfer within the enzyme, and not, as previously suggested, to saturation of substrate binding to the enzyme. Also, the term k0[S] implies that the reactive substrates, including the natural substrate (yeast cytochrome c), react with the hydrogen peroxide-heme complex and not with the radical species formed by reaction with hydrogen peroxide in the absence of reductants.     

Electron paramagnetic and electron nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase

We have collected electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectra from the hydrogen peroxide compound of yeast cytochrome c peroxidase, termed ES, employing EPR microwave frequencies of 9.6 and 11.6 GHz. We have measured and analyzed the temperature dependence of the spin-lattice relaxation rate (1/T1) of the paramagnetic center of ES over the temperature range 1.9 to 4 K. In addition, an upper bound to exchange coupling between the ferryl heme and EPR-visible centers of ES has been calculated and expressions for the dipolar interaction between a ferryl heme and a free radical have been derived. These results all confirm that the EPR signal of ES is not associated with an aromatic amino acid radical, and in particular not with a tryptophanyl radical. This conclusion has led us to consider an explanation of the EPR signal in terms of a nucleophilically stabilized methionyl radical.     

Engineering ascorbate peroxidase activity into cytochrome c peroxidase

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each other's activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate.     

The degradation of cytochrome c by hydrogen peroxide

Cytochrome c is degraded by a large excess of hydrogen peroxide, leading to opening of the heme porphyrin ring and loss of the Soret absorption bands. The kinetic parameters of this reaction have been determined, and it is shown that a small concentration of oxygen is liberated at the same rate as degradation. Low-level chemiluminescence and release of a hydroxylating species also accompany heme destruction. It is proposed that heme iron activates hydrogen peroxide to a more powerful oxidant, perhaps the hydroxyl radical, which remains bound to the heme iron and initiates attack on the porphyrin ring. Chemiluminescence appears to result from a side reaction involving singlet oxygen attack on the alpha-methene bridge, yielding a dioxetane. The in vivo degradation of cytochrome c by excess hydrogen peroxide may interfere with respiration, accelerate aging, and enhance the metabolism of carcinogens.     

Oxidative modification of cytochrome c by hydrogen peroxide

Oxidative alteration of mitochondrial cytochrome c has been linked to disease and is one of the causes of pro-apoptotic events. We have investigated the modification of cytochrome c by H2O2. When cytochrome c was incubated with H2O2, oligomerization of the protein increased and the formation of carbonyl derivatives and dityrosine was stimulated. Radical scavengers prevented these effects suggesting that free radicals are implicated in the H2O2-mediated oligomerization. Oligomerization was significantly inhibited by the iron chelator, deferoxamine. During incubation of deoxyribose with cytochrome c and H2O2, damage to the deoxyribose occurred in parallel with the release of iron from cytochrome c. When cytochrome c that had been exposed to H2O2 was analyzed by amino acid analysis, the tyrosine, histidine and methionine residues proved to be particularly sensitive. These results suggest that H2O2-mediated cytochrome c oligomerization is due to oxidative damage resulting from free radicals generated by a combination of the peroxidase activity of cytochrome c and the Fenton reaction of free iron released from the oxidatively-damaged protein.     

Complex-formation between cytochrome c and cytochrome c peroxidase. Equilibrium and titration studies

1. Physical studies of complex-formation between cytochrome c and yeast peroxidase are consistent with kinetic predictions that these complexes participate in the catalytic activity of yeast peroxidase towards ferrocytochrome c. Enzyme-ferricytochrome c complexes have been detected both by the analytical ultracentrifuge and by column chromatography, whereas an enzyme-ferrocytochrome c complex was demonstrated by column chromatography. Estimated binding constants obtained from chromatographic experiments were similar to the measured kinetic values. 2. The physicochemical study of the enzyme-ferricytochrome c complex, and an analysis of its spectrum and reactivity, suggest that the conformation and reactivity of neither cytochrome c nor yeast peroxidase are grossly modified in the complex. 3. The peroxide compound of yeast cytochrome c peroxidase was found to have two oxidizing equivalents accessible to cytochrome c but only one readily accessible to ferrocyanide. Several types of peroxide compound, differing in available oxidizing equivalents and in reactivity with cytochrome c, seem to be formed by stoicheiometric amounts of hydrogen peroxide. 4. Fluoride combines not only with free yeast peroxidase but also with peroxidase-peroxide and accelerates the decomposition of the latter compound. The ligand-catalysed decomposition provides evidence for one-electron reduction pathways in yeast peroxidase, and the reversible binding of fluoride casts doubt upon the concept that the peroxidase-peroxide intermediate is any form of peroxide complex. 5. A mechanism for cytochrome c oxidation is proposed involving the successive reaction of two reversibly bound molecules of cytochrome c with oxidizing equivalents associated with the enzyme protein.     

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).