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.     

Characterization of a covalently linked yeast cytochrome c-cytochrome c peroxidase complex: evidence for a single, catalytically active cytochrome c binding site on cytochrome c peroxidase

A covalent complex between recombinant yeast iso-1-cytochrome c and recombinant yeast cytochrome c peroxidase (rCcP), in which the crystallographically defined cytochrome c binding site [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] is blocked, was synthesized via disulfide bond formation using specifically engineered cysteine residues in both yeast iso-1-cytochrome c and yeast cytochrome c peroxidase [Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580]. Previous studies on similar covalent complexes, those that block the Pelletier-Kraut crystallographic site, have demonstrated that samples of the covalent complexes have detectable activities that are significantly lower than those of wild-type yCcP, usually in the range of approximately 1-7% of that of the wild-type enzyme. Using gradient elution procedures in the purification of the engineered peroxidase, cytochrome c, and covalent complex, along with activity measurements during the purification steps, we demonstrate that the residual activity associated with the purified covalent complex is due to unreacted CcP that copurifies with the covalent complex. Within experimental error, the covalent complex that blocks the Pelletier-Kraut site has zero catalytic activity in the steady-state oxidation of exogenous yeast iso-1-ferrocytochrome c by hydrogen peroxide, demonstrating that only ferrocytochrome c bound at the Pelletier-Kraut site is oxidized during catalytic turnover.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse 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.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

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 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.     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.     

Effects of surface amino acid replacements in cytochrome c peroxidase on complex formation with cytochrome c.

Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase. The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins. The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography. The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme. The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase. The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c. This empirical finding localizes a primary recognition region critical to the dynamic complex. Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.     

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.     

Cytochrome c peroxidase catalyzed oxidation of ferrocytochrome c by hydrogen peroxide: ionic strength dependence of the steady-state rate parameters

The steady-state kinetics of the cytochrome c peroxidase catalyzed oxidation of horse heart ferrocytochrome c by hydrogen peroxide have been studied at both pH 7.0 and pH 7.5 as a function of ionic strength. Plots of the initial velocity versus hydrogen peroxide concentration at fixed cytochrome c are hyperbolic. The limiting slope at low hydrogen peroxide give apparent bimolecular rate constants for the cytochrome c peroxidase-hydrogen peroxide reaction identical with those determined directly by stopped-flow techniques. Plots of the initial velocity versus cytochrome c concentration at saturating hydrogen peroxide (200 microM) are nonhyperbolic. The rate expression requires squared terms in cytochrome c concentration. The maximum turnover rate of the enzyme is independent of ionic strength, with values of 470 +/- 50 s-1 and 290 +/- 30 s-1 at pH 7.0 and 7.5, respectively. The limiting slope of velocity versus cytochrome c concentration plots provides a lower limit for the association rate constant between cytochrome c and the oxidized intermediates of cytochrome c peroxidase. The limiting slope varies from 10(6) M-1 s-1 at 300 mM ionic strength to 10(8) M-1 s-1 at 20 mM ionic strength and extrapolates to 5 x 10(8) M-1 s-1 at zero ionic strength. The data are discussed in terms of both a two-binding-site mechanism and a single-binding-site, multiple-pathway mechanism.     

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

Cytochrome c/cytochrome c oxidase interaction. Direct structural evidence for conformational changes during enzyme turnover.

We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the 'traditional' oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady-state reducing conditions. Whereas for the 'oxidized' complex cytochrome c became bound exclusively to subunit II of the enzyme, for the 'steady-state' complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV. For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Steady-state spectral analysis (400-630 nm) combined with stopped-flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer. Additionally, stopped-flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c: The second order rate constant for the traditional complex was 0.55x10(6) m(-1) x s(-1) while that for the steady-state was 0.27x10(6) m(-1) x s(-1). These values were approximately 25-50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.     

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.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.     

Reaction of cytochrome c with nitrite and nitric oxide. A model of dissimilatory nitrite reductase.

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.     

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.