Lidia B. Vitello

Yeast cytochrome c peroxidase: mechanistic studies via protein engineering

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water by ferrocytochrome c. It was the first heme enzyme to have its crystallographic structure determined and, as a consequence, has played a pivotal role in developing ideas about structural control of heme protein reactivity. Genetic engineering of the active site of CcP, along with structural, spectroscopic, and kinetic characterization of the mutant proteins has provided considerable insight into the mechanism of hydrogen peroxide activation, oxygen-oxygen bond cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The catalytic mechanism involves complex formation between cytochrome c and CcP. The cytochrome c/CcP system has been very useful in elucidating the complexities of long-range electron transfer in biological systems, including protein-protein recognition, complex formation, and intracomplex electron transfer processes.

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: A fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.

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 degrees 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 1:1 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.

Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants.

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.

Sedimentation equilibrium studies on the interaction between cytochrome c and cytochrome c peroxidase

The interaction between cytochrome c and cytochrome c peroxidase was investigated using sedimentation equilibrium at pH 6,20 degrees C, in a number of buffer systems varying in ionic strength between 1 and 100 mM. Between 10 and 100 mM ionic strengths, the sedimentation of the individual proteins was essentially ideal, and sedimentation equilibrium experiments on mixtures of the two proteins were analyzed assuming ideal solution behavior. Analysis of the distribution of mixtures of cytochrome c and cytochrome c peroxidase in the ultracentrifuge cell based on a model involving the formation of a 1:1 cytochrome c-cytochrome c peroxidase complex gave values of the equilibrium dissociation constant ranging from 2.3 +/- 2.7 microM at 10 mM ionic strength to infinity (no detectable interaction) at 100 mM ionic strength. Attempts to determine the presence of complexes involving two cytochrome c molecules bound to cytochrome c peroxidase were inconclusive.

Electron Transfer within the Cytochrome c-Cytochrome c Peroxidase 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.

Apolar Distal Pocket Mutants of Yeast Cytochrome c Peroxidase: Hydrogen Peroxide Reactivity and Cyanide Binding of the TriAla, TriVal, and TriLeu Variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H(2)O(2) compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H(2)O(2) and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H(2)O(2) and HCN while features that inhibit substrate ionization slow the reactions.

Block poly(Ala)-poly(Lys). A water-soluble model for intrinsic membrane proteins?

Block poly(Ala)16-poly(Lys)13.5 was synthesized by the Leuchs anhydride method. This polypeptide is water soluble in a largely monomeric form, but binds rapidly and spontaneously to unilamellar vesicles of dimyristoyl phosphatidylcholine at pH 7.4. The interaction is evidently of a hydrophobic nature since the complex is not disrupted by salt and no similar reaction is given by polylysine. Evidence for the interaction was obtained by ultrafiltration, chromatography on Sepharose 4B, and sedimentation velocity ultracentrifugation. While direct information on the molecular structure of the complex is still lacking, we propose that this amphipathic block copolymer binds to lipids in a similar manner as intrinsic membrane proteins and hence can be used to study the interactions of intrinsic proteins with lipids.

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II

The bis(terpyridine)cobalt(II), Co(terpy)22+, reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 °C. Co(terpy)22+ initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0±0.4×106 M–1s–1 at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s–1 was observed for the reduction of CcP-I by 79 µM Co(terpy)22+ at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)22+ shows saturation kinetics with a maximum observed rate constant, k3max, of 24±2 s–1 at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)22+ reduction of CcP-II, k3, is 1.2±0.5×106 M–1 s–1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0±0.8×105M–1 s–1, 1.2±0.5×105M–1 s–1, and 11±3 s–1, respectively. Both the product, Co(terpy)23+, and ferricytochrome c inhibit the rate of Co(terpy)22+ reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)23+ molecules bind to the native enzyme in low ionic strength buffers.

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 (KD(app)) 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 KD(app) 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.