Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

A stopped-flow study of the reaction of cytochrome c peroxidase with hydroperoxides

Transient kinetic measurements show that cytochrome c peroxidase reacts with excess of hydroperoxides to produce compound ES in two phases. The activation energies for the fast and slow phases are calculated to be 6.3 and 20.5 kcal X mol-1, respectively. The fast phase is assigned to the reaction of native active (pulsed) cytochrome c peroxidase with peroxides, whereas the slow phase is due to the presence of an inactive (aged, resting) enzyme. As the active species is exhausted, the equilibrium between the active and inactive enzymes is shifted by a slow conformational change to replenish the active enzyme. Since the rate-limiting step of the reaction of the inactive enzyme with peroxides is the conformation change, the overall reaction rate is independent of the nature and concentration of peroxides.     

Crystal structure of cytochrome c peroxidase compound I

We have compared the 2.5-A crystal structure of yeast cytochrome c peroxidase (CCP) with that of its semistable two-equivalent oxidized intermediate, compound I, by difference Fourier and least-squares refinement methods. Both structures were observed at -15 degrees C. The difference Fourier map reveals that formation of compound I causes only small positional adjustments of a few tenths of an angstrom. The map's most pronounced feature is a pair of positive and negative peaks bracketing the heme iron position. Least-squares refinement shows that the iron atom moves about 0.2 A toward the distal side of the heme. No significant difference density is evident near the side chains of Trp-51 or Met-172, each of which has been proposed to be the site of the electron paramagnetic resonance (EPR) active radical in compound I. However, the second most prominent feature of difference density is a negative peak near the side chain of Thr-180, which, according to the results of least-squares refinement, moves by 0.15 A in the direction of Met-230. These observations, together with the results of mutagenesis experiments [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360; Goodin, D. B., Mauk, A. G., & Smith, M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1295-1299] in which Trp-51 and Met-172 have been replaced without loss of the EPR radical signal in compound I, lead us to consider the possibility that the radical site lies within a cluster composed of the side chains of Met-230, Met-231, and Trp-191.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution crystal structures and spectroscopy of native and compound I cytochrome c peroxidase

Cytochrome c peroxidase (CCP) is a 32.5 kDa mitochondrial intermembrane space heme peroxidase from Saccharomyces cerevisiae that reduces H(2)O(2) to 2H(2)O by oxidizing two molecules of cytochrome c (cyt c). Here we compare the 1.2 A native structure (CCP) with the 1.3 A structure of its stable oxidized reaction intermediate, Compound I (CCP1). In addition, crystals were analyzed by UV-vis absorption and electron paramagnetic resonance spectroscopies before and after data collection to determine the state of the Fe(IV) center and the cationic Trp191 radical formed in Compound I. The results show that X-ray exposure does not lead to reduction of Fe(IV) and only partial reduction of the Trp radical. A comparison of the two structures reveals subtle but important conformational changes that aid in the stabilization of the Trp191 cationic radical in Compound I. The higher-resolution data also enable a more accurate determination of changes in heme parameters. Most importantly, when one goes from resting state Fe(III) to Compound I, the His-Fe bond distance increases, the iron moves into the porphyrin plane leading to shorter pyrrole N-Fe bonds, and the Fe(IV)-O bond distance is 1.87 A, suggesting a single Fe(IV)-O bond and not the generally accepted double bond.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

The primary structure of Pseudomonas cytochrome c peroxidase

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met-His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated M_r of 33 690.     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.     

Structural and functional features of Pseudomonas cytochrome c peroxidase.

The secondary structure of Pseudomonas cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) has been predicted from the established amino acid sequence of the enzyme using a Chou-Fasman-type algorithm. The amount of alpha-helicity thus obtained is in agreement with previously obtained results based on circular dichroic measurements at far UV. The two heme c moieties of the enzyme have earlier been shown to have widely different characteristics, e.g., the redox potentials of the hemes differ with about 600 mV, and carry out different functions in the enzyme molecule. The structural comparisons made in this study enlighten the observed functional differences. The first heme in the polypeptide chain, heme 1, has in its environment a folding pattern generally encountered in cytochromes. In the region of the sixth ligand, however, profound differences are noted. The cytochromal methionine has been replaced by a lysine with a concomitant lowering of redox-potential thus making peroxidatic activity possible. Around heme 2, extra amino acid residues have been added to the peroxidase as compared with Rhodospirillum molischianum cytochrome c2 core structure in the 20's loop. After completion of the cytochromal fold around heme 2 an additional tail consisting of 25 residues is linked. This tail shows no stabilizing elements of secondary structure, but contains a strongly hydrophobic segment which suggests a possible membrane contact site of this extrinsic membrane protein. Heme 2 is concluded to have a cytochromal function in the molecule. To further elucidate the functional properties of the enzyme, a noncovalent two-fragment complex was produced by specific cleavage of the peroxidase by Pseudomonas elastase. The complex was studied with respect to its properties to the native enzyme. The two-fragment complex of Pseudomonas peroxidase retains the overall conformation of the native enzyme showing, however, no heme-heme interaction. Thus, a comparison of the properties of the native enzyme with those of the two-fragment complex permitted some conclusions to be drawn on the structure of the enzyme as well as the mechanism of heme-heme interaction. From the present results we conclude that the two distal heme surfaces in the peroxidase are oriented toward each other. This structural arrangement allows an inter-heme communication in the enzyme molecule and it also forms the structural basis for the enzyme mechanism. The structural comparisons also give insight into the evolution of an ancestral cytochrome c into an efficient peroxidase that has a versatile control mechanism in heme-heme interaction.     

pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase.

A pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase was carried out at 25 degrees C and 0.1 M ionic strength. The net charge on cytochrome c peroxidase due to proton association and dissociation varies from +32 at pH 2 to --50.2 at pH 12, while that of apocytochrome c peroxidase varies between +24.5 at pH 3 to --48 at pH 12. The apoprotein tented to aggregate below pH 3. Between pH 4 and 8, the titration behavior of both the native enzyme and the apoenzyme are consistent with the semi-empirical Linderstr?m-Lang theory. Between pH 9 and 12, the titration behavior of both the holo- and apoproteins suggest they assume a more extended conformation which reduces the electrostatic interaction charged groups on the surface. In the acid region, between pH 4 and 3, a similar transition occurs in which the protein expands 40% based on the electrostatic factor of the Linderstr?m-Lang theory.     

Ligand binding to cytochrome c peroxidase from Pseudomonas aeruginosa

The high potential heme site of Pseudomonas cytochrome c peroxidase has His and Met as ligands. On reduction, the Fe-met bond becomes photosensitive. Following photolysis, the bond reforms with a half-time of 35 ps. The low potential heme peroxidatic site of the fully reduced enzyme has been shown to bind to a range of ligands. The compounds with carbon monoxide, methyl, ethyl, n-butyl, and t-butyl isonitriles have been investigated by laser flash photolysis. All are photosensitive and show different degrees of geminate recombination of ligand in the picosecond and nanosecond time ranges. Carbon monoxide shows the least effect. The three straight-chain isonitriles show about 50% geminate recombination with half-times of the order of 10 ns. t-Butyl isonitrile shows more and faster recombination. These results imply considerable freedom of movement within the active site for the smaller ligands.     

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (R?nnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.     

M?ssbauer spectroscopic study of compound ES of cytochrome c peroxidase.

M?ssbauer spectra of Compound ES of cytochrome c peroxidase have been observed over a range of temperature and applied magnetic field. These have been interpreted in terms of a crystal field model of the iron site in which the iron is assumed to be in the Fe(IV) state with unpaired spin S = 1. Detailed least-squares fitting of the spectra fitting of the spectra indicates that both the electric field gradient choice of a single parameter, the axial crystal field, the magnetic properties are well reproduced. The model also provides the observed positive sign for the electric field gradient interaction, but overestimates its magnitude. This apparent discrepnancy may be caused by the presence of significant electronic charge in filled bonding orbitals, a feature which is in keeping with expected covalent charge compensation of the extreme oxidation state. There is no evidence in the M?ssbauer spectra of interaction between the iron and the ESR-visible free radical. This suggests they are well separated.     

Isolation and properties of cytochrome c peroxidase from Pseudomonas denitrificans.

The isolation of cytochrome c peroxidase, cytochrome c4, cytochrome c-551 and azurin from Pseudomonas dentrificans is described. The peroxidase has a molecular weight of 63,000 and an isoelectric point of 5.6. Its absorption spectrum suggests that it contains two haem c groups/molecule. Preliminary steady-state kinetic data are reported with cytochromes c-551 and c4 and azurin as the second substrate.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc–CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.     

Oxidation of horseradish peroxidase compound II to compound I.

In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH.