Proton NMR comparison of the Saccharomyces cerevisiae ferricytochrome c isozyme-1 monomer and covalent disulfide dimer

Proton NMR studies of Saccharomyces cerevisiae (bakers yeast) isozyme-1 monomer and dimer ferricytochrome c have been carried out. The dimer is formed via a disulfide bridge between the Cys-102 residues of monomer proteins. Nuclear Overhauser effect (NOE) experiments have led to resonance assignments for many of the heme and axial ligand (Met-80; His-18) protons in both protein forms. Resonances of the following amino acids have also been assigned in both forms: Phe-10; Pro-30; Phe-82; Trp-59; Leu-68. The proton NOE connectivity patterns of the monomer of yeast isozyme-1 ferricytochrome c are similar to those of horse, tuna, and yeast isozyme-2 ferricytochromes c, even though the observed hyperfine resonance spectra are significantly different for the various cytochromes. The pattern of dimer proton hyperfine resonances is distinct from the isozyme-1 monomer pattern, which indicates that the formation of a disulfide bridge via Cys-102 is detected at the heme site, approximately 10 A distant. It appears that a specific structural change is induced upon dimerization, which, in turn, causes specific perturbations in the vicinity of the heme. However, the general features of the NOE connectivity pattern in the dimer are the same as for the monomer indicating that dimerization does not result in drastic structural disruption. Furthermore, the 1H NMR spectrum of the dimer can be mimicked by the monomer form that results when the -SH group of Cys-102 is chemically modified with certain types of bulky, or hydrophilic reagents (i.e. 5,5'-dithiobis[2-nitrobenzoate], indicating that perturbations of the yeast isozyme-1 ferricytochrome c proton resonance spectrum observed upon dimerization are essentially due to changes in intramolecular, rather than intermolecular, interactions. These results suggest that a possible regulatory site for yeast isozyme-1 cytochrome c exists at position 102, which could conceivably have a physiological role in altering the conformation of the molecule.     

One- and two-dimensional proton NMR studies of cys-102 S-methylated yeast isozyme-1 ferricytochrome c.

The effect of S-methylating cysteine-102 (cys-102) (SH----SSCH3) of yeast isozyme-1 (iso-1) ferricytochrome c has been studied using proton NMR spectroscopy. COSY, NOESY, and one-dimensional nuclear Overhauser effect (NOE) difference spectroscopies have all been used. The NMR spectrum of this derivative is very similar to that of native yeast iso-1 ferricytochrome c. The advantage of using the cys-102 S-methylated derivative is that it is unable to spontaneously dimerize in solution, like native iso-1 monomer does. This makes the derivative a simple, ideal protein for long NMR experiments. This work yields many proton resonance assignments for S-methylated yeast iso-1 monomer and confirms all of the assignments for iso-1 monomer that were previously made using only the one-dimensional NOE method.     

Solution structure of mitochondrial cytochrome c. I. 1H nuclear magnetic resonance of ferricytochrome c

The 1H nuclear magnetic resonance spectra of tuna and horse ferricytochromes c have been investigated and the resonances of all amino acid methyl groups have been assigned to specific absorption lines. The assignment procedure involves principally the comparison of one-dimensional nuclear magnetic resonance spectra from a range of homologous ferricytochromes c and does not require a prior knowledge of the secondary or tertiary protein structure. Of the 49 methyl groups of tuna cytochrome c, the assignment of 33 is made without reference to the X-ray crystal structure. The method should therefore be applicable to other proteins of similar size where X-ray structures are unavailable. The assignments will be used to investigate the structure of cytochrome c in solution.     

The solution structures of tuna and horse cytochromes c

The nuclear magnetic resonance spectra of tuna ferricytochrome c and tuna ferrocytochrome c are described. Resonance assignments are made using NMR double-resonance techniques. A comparison of the NMR data for tuna cytochrome c with the previously reported data for horse cytochrome c shows that the proteins have virtually identical main-chain folds. Three regions of local conformational differences have been distinguished.     

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide

The 1:1 covalently cross-linked complex between horse cytochrome c and yeast cytochrome c peroxidase (ccp) has been formed by a slight modification of the method of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184-5190]. This earlier study has been extended to show that efficient cross-linking of the two proteins can occur in a variety of buffers over a broad ionic strength range. The substitution of ferrocytochrome c for ferricytochrome c in the cross-linking studies resulted in an increased yield of 1:1 complex (approximately 10-20%) under the conditions studied. An improved method for purifying the covalent complex in relatively large quantities is presented here as are the results of electrophoresis and proton NMR studies of the complex. Both electrophoresis and NMR studies indicate modification of some surface acidic amino acids in the covalent complex by the carbodiimide. The proton hyperfine-shifted resonances of cytochrome c are broadened in the covalent complex relative to free cytochrome c, and the resonances corresponding to the cytochrome c heme 3-CH3 and 8-CH3 groups are shifted closer together in the complex. Integration of NMR resonances confirms a 1:1 complex as the primary cross-linking reaction product. However, we also demonstrate that the covalent complex can be further coupled to ccp and to cytochrome c to form higher molecular weight aggregates.     

Species-specific differences in covalently crosslinked complexes of yeast cytochrome C peroxidase with horse and yeast ISO-1 ferricytochromes C

• 1.1. The results of chemically crosslinking yeast cytochrome c peroxidase with both horse and yeast iso-1 ferricytochromes c have been studied by a combination of gel electrophoresis and proton NMR spectroscopy.
• 2.2. The complexes were formed at a variety of potassium phosphate concentrations ranging from 10 to 300 mM using the water soluble crosslinking agent, EDC (l-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide).
• 3.3. The primary crosslinking product in both cases is the 1:1 covalent complex, but, for each pair of partner proteins the yield of the 1:1 crosslinked complex varies with the salt concentration.
• 4.4. Furthermore, at low salt concentrations the yield of the 1:1 covalent complex involving horse cytochrome c is much larger than the yield of the 1:1 covalent complex formed with yeast iso-1 cytochrome c, whereas at high salt concentrations the situation is reversed.
• 5.5. Proton NMR spectroscopy, in combination with gel electrophoresis, provides evidence for the formation of different types of 1:1 complexes for the peroxidase/yeast cytochrome c pair and has been used to study the effect of changes in the solution ionic strength upon both the peroxidases/horse cytochrome c and the peroxidase/yeast cytochrome c complexes.
• 6.6. This work indicates that electrostatic interactions between proteins play a dominant role in formation of complexes between cytochrome c peroxidase and horse ferricytochrome c, whereas the hydrophobic effect plays a comparatively larger role in stabilizing complexes between cytochrome c peroxidase and yeast iso-1 ferricytochrome c.

     

Fourier-transform infra-red studies of the alkaline isomerization of mitochondrial cytochrome c and the ionization of carboxylic acids.

The alkaline transitions of tuna and horse ferricytochromes c and the trifluoroacetyl-lysine derivative of horse ferricytochrome c have been studied by Fourier-transform (FT) i.r. spectroscopy. The spectral perturbations resulting from the transition have been interpreted by reference to FT i.r. data on simple carboxylic-acid-containing compounds and a bacterial cytochrome c551 in which a haem propionate ionizes without causing a significant conformational change. The analysis strongly suggests that ionization of a haem propionate of mitochondrial cytochrome c triggers the alkaline conformation change.     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes

Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and horse cytochrome c, but different from those reported for cytochrome c complexes with flavodoxin and cytochrome b5. By comparison with the shifts reported for lysine-13-modified cytochrome c we conclude that the results reported here support the Poulos-Kraut proposed structure for the molecular redox complex between cytochrome-c peroxidase and cytochrome c. These results indicate that the principal site of interaction with cytochrome-c peroxidase is the exposed heme edge of horse cytochrome c, in proximity to lysine-13 and the heme pyrrole II. The noncovalent cytochrome-c peroxidase-cytochrome c complex exists in the rapid-exchange time limit even at 500 mHz proton frequency. Our data provide an improved estimate of the minimum off-rate for exchanging cytochrome c as 1133 (+/- 120) s-1 at 23 degrees C.     

Study of the biological significance of cytochrome methylation. I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c.

The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.     

Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.     

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

Comparison of the structures of various eukaryotic ferricytochromes c and ferrocytochromes and their antigenic differences

The nuclear magnetic resonance spectra of various eukaryotic ferricytochromes c and ferrocytochromes c are described. The proteins from the species donkey, cow, dog, rabbit, chicken and pigeon were investigated. The conformations of these proteins detected by NMR were compared to those of horse and tuna cytochromes c and in some cases small differences were found. These differences in structure were shown to correlate with antigenic differences between the various proteins.     

Kinetics of electron transfer between mitochondrial cytochrome c and iron hexacyanides

The reduction of horse and Candida krusei cytochromes c by ferrocyanide has been studied by 1H NMR spectroscopy and the reaction found to involve a precursor complex of ferrocyanide bound to ferricytochrome c (pH* 7.4, 2H2O, I = 0.12, and 25 degrees C). The electron transfer rate constants for the reduction of the two ferricytochromes by associated ferrocyanide were found to be the same at 780 +/- 80 sec-1 but the association constants for binding of ferrocyanide to ferricytochrome c were significantly different: horse, 90 +/- 20 M-1 and Candida, 285 +/- 30 M-1. The different association constants partly accounts for the previously observed reactivity difference between horse and Candida cytochromes c. Comparison of the NMR data with data obtained by other kinetic methods has allowed the electron transfer rate constant for the oxidation of ferrocytochrome c by associated ferricyanide to be determined. This was found to be 4.6 +/- 1 X 10(4) sec-1.     

Interactions between yeast iso-1-cytochrome c and its peroxidase

Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP. The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution. Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex. Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex.     

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.     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aliphatic amino acids

The aliphatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. There are eight firmly assigned methyl resonances of ferrocytochrome c and seven firmly assigned methyl resonances of ferricytochrome c.     

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.