Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

Derivatives of yeast iso-1 cytochrome c, chemically modified at Cys-102 (Cys-102 acetamide-derivatized monomer, Cys-102 thionitrobenzoate-derivatized monomer, Cys-102 S-methylated monomer, and the disulfide dimer), exhibit different spectral and physicochemical properties relative to the native, unmodified protein, depending on the nature of the modifying group. The results of proton NMR studies on the Cys-102 acetamidederivatized monomer of iso-1 ferricytochrome c indicate that the conformational characteristics of the heme environment in this protein derivative are intermediate between those of the unmodified monomer and disulfide dimer forms of the protein. Measurements of the pK_a of the alkaline transitions of the five forms of iso-1 ferricytochrome c provided values of 8.89, 8.82, 8.67, 8.47, and 8.50 for the unmodified monomer, S-methylated monomer, acetamide-derivatized monomer, thionitrobenzoate-derivatized monomer, and disulfide dimer, respectively. The results of proton NMR studies of the reduced form of these proteins suggest that the heme environments of the unmodified monomer and disulfide dimer derivatives of iso-1 ferrocytochrome c are similar and indicate that treatment of the thionitrobenzoate-derivatized and disulfide dimer forms of the protein with sodium dithionite results in cleavage of the disulfide bonds at position 102. Circular dichroism studies reveal that only the disulfide dimer form of iso-1 ferricytochrome c exhibits a Soret CD spectrum which differs from the native, unmodified monomer in that the intensity of the negative band at approximately 420 nm is diminished in the spectrum of the dimer relative to the spectrum of the monomer. Soret CD spectra of the ascorbate-reduced form of all protein derivatives are similar. The process of autoreduction of yeast iso-1 ferricytochrome c is shown to occur in the absence of a free sulfhydryl group at position 102 and is exacerbated under moderately high pH conditions. These results are suggestive of the presence of a redox-active amino acid, perhaps a tyrosine, in yeast iso-1 cytochrome c.     

pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.     

Refolding a disulfide dimer of cytochrome c

A covalent dimer of Saccharomyces cerevisiae iso-1 cytochrome c is stabilized by an interchain disulfide bond involving the cysteine residue penultimate to the C-terminus. The individual chains in the dimer appear to retain the tertiary structural features characteristic for monomeric cytochrome c albeit with some perturbation. The dimer is reversibly denatured by heat, urea, or guanidine hydrochloride in a single cooperative transition whose midpoint is less than that of the monomeric protein. The kinetic profile observed for the refolding of the denatured dimer is characteristic for monomeric cytochromes except for a markedly enhanced slow-phase amplitude.     

Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases

The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.     

Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c

As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Replacement of a conserved proline and the alkaline conformational change in iso-2-cytochrome c

Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change. The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered heme ligation. To assess the importance of position 76 in stabilizing the native versus the alkaline structure, the equilibrium and kinetic properties of the pH-induced conformational change have been compared for normal and mutant iso-2-cytochrome c. The pKapp for the conformational change is reduced from 8.45 (normal iso-2) to 6.71 in the mutant protein (Gly-76 iso-2), suggesting that conservation of Pro-76 may be required to stabilize the native conformation at physiological pH. The kinetics of the conformational change for both the normal and mutant proteins are well-described by a single kinetic phase throughout most of the pH-induced transition zone. Over this pH range, a minimal mechanism proposed for horse cytochrome c [Davis, L. A., Schejter, A., & Hess, G. P. (1974) J. Biol. Chem. 249, 2624-2632] is consistent with the data for normal and mutant yeast iso-2-cytochromes c: NH KH----N + H+ kcf in equilibrium kcb A NH and N are native forms of cytochrome c with a 695-nm absorbance band, A is an alkaline form that lacks the 695-nm band, KH is a proton dissociation constant, and kcf and kcb are microscopic rate constants for the conformational change. The Gly-76 mutation increases kcf by almost 70-fold, but kcb and KH are unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 conformational transition of horse heart porphyrin c

The heme iron of horse heart cytochrome c was selectively removed using anhydrous HF. The product, porphyrin c, exhibits the viscosity, far ultraviolet circular dichroic, and fluorescence properties characteristic for native cytochrome c. However, porphyrin c is more susceptible to denaturation by guanidine hydrochloride and by heat than is the parent cytochrome. All of the conformational parameters of porphyrin c exhibit a common reversible transition centered at 0.95 m guanidine hydrochloride at 23 degrees C and pH 7.0. Guanidine denatured porphyrin c refolds in two kinetic phases having time constants of 20 and 200 ms as detected by stopped flow absorbance or fluorescence measurement, with about 80% of the observed change in the faster phase. The kinetics of porphyrin c refolding are not significantly altered by increasing the viscosity of the refolding solvent 15-fold by addition of sucrose. We suggest that the folding of guanidine denatured cytochrome c is not a diffusion-limited process and that the requirement for protein axial ligation elicits the slow (s) kinetic phase observed in the refolding of cytochrome c.     

Effective concentrations of amino acid side chains in an unfolded protein.

Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Many faces of the unfolded state: conformational heterogeneity in denatured yeast cytochrome C

We have measured fluorescence energy-transfer (FET) kinetics from a dansyl fluorophore (Dns) introduced by derivatization of a Cys side-chain to the Fe(III) heme covalently attached to unfolded yeast iso-1 cytochrome c (cyt). To gain a global picture of the unfolded state, we examined variants with the fluorophore attached on three different helices (K4C, E66C, K99C) and in three different loops (H39C, D50C, L85C). Analysis of the FET kinetics data gave distributions of distances between the fluorescent donor and acceptor; these distributions demonstrate that the guanidine hydrochloride (GuHCl)-denatured polypeptide ensemble is not a simple random coil. Although misligation imposes some constraints, it is not the only source of structural complexity in the unfolded protein. Our FET kinetics data reveal a high degree of heterogeneity in the unfolded ensemble of cytochrome c. We detect relatively large populations of compact structures in unfolded Dns(C50)cyt, Dns(C39)cyt, and Dns(C66)cyt. These structures likely play a role in forming a hydrophobic core during the folding process.     

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.     

Conservation of the free energy change of the alkaline isomerization in mitochondrial and bacterial cytochromes c

The thermodynamic parameters of the alkaline transition for oxidized native yeast iso-1 cytochrome c and Rhodopseudomonas palustris cytochrome c(2) (cytc(2)) have been determined through direct electrochemistry experiments carried out at variable pH and temperature and compared to those for horse and beef heart cytochromes c. We have found that both transition enthalpy and entropy are remarkably species dependent, following the order R. palustris cytc(2) > beef (horse) heart cytc>yeast iso-1 cytc. Considering the high homology at the heme-protein interface in the native species, this variability is likely to be mainly determined by differences in the structural and solvation properties and the relative abundance of the various alkaline conformers. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol(-1)) and consequently in the apparent pK(a) value. This compensation indicates that solvent reorganization effects play an important role in the thermodynamics of the transition. This mechanism is functional to ensure a relatively high pK(a) value for the alkaline transition, which is needed to preserve His,Met ligation to the heme iron in cytochrome c at physiological pH and temperature, hence the E(o) value required for the biological function.     

Ubiquitin conjugation to cytochromes c. Structure of the yeast iso-1 conjugate and possible recognition determinants.

Saccharomyces cerevisiae iso-1-cytochrome c was conjugated with ubiquitin (Ub) in vitro in a rabbit reticulocyte extract (Fraction II). By N-terminal protein sequencing, it was found for both the mono- and diubiquitinated products that the major Ub attachment site is on Lys4 (residue 9) of the cytochrome c. Thus, the residue ubiquitinated in iso-1-cytochrome c is identical with that previously determined for the yeast iso-2 form (Sokolik, C. W., and Cohen, R. E. (1991) J. Biol. Chem. 266, 9100-9107). For both cytochromes c, the proportions of diubiquitinated and higher order conjugates are drastically reduced when Ub is replaced with a Lys48----Arg variant, suggesting that the Ub-Ub moieties are linked predominantly through Lys48. Despite close similarities in structure and ubiquitination sites, conjugation to iso-2-cytochrome c is approximately 5-fold faster than for the iso-1 form; vertebrate cytochromes c are even poorer substrates, being ubiquitinated at only approximately 5% of the rate of the iso-2 protein. Comparison of several cytochrome c variants excludes alpha-N-acetylation or the identity of the N-terminal amino acid as the important recognition determinants in these reactions. The results, which include the finding that ferro and ferri-iso-2-cytochromes c are ubiquitinated equally, also are evidence against a simple correlation between ubiquitination efficiency and thermodynamic stability. Rather, the presence of a pair of lysines (Lys4-Lys5) within the relatively unstructured N-terminal extension of the yeast cytochromes c may be responsible for their preferential ubiquitination.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.

1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase.