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 K,, = 5 x 1Om8 M, and apparent K,, = 0.35 to 1.0 x 10m6 M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with K,, I lo-’ M, and KD, = 1O-6 M. 2. The maximal velocities at low ionic strength increased with pH and were highest above pH 7.5. 3. The presence and properties of the low apparent K, 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 > ADP >> P, 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. 4. At low concentrations bakers’ yeast iso-1,

and sodium cyanide (0.25 rn~) for 3 min in the same buffer as for the polarographic assay to obtain complete inhibition of the oxidase activity.
The initial rate of reduction was measured at 550 nm after addition of cytochrome c (0.3 to 7 PM), using a reduced minus oxidized millimolar extinction coefficient of 18.7. Binding of Ferricytochrome c to Cytochrome c Oridase-This binding was measured using gel filtration, as described for this system by C. R. Hartzella and Hartzell and Shaw (43). Samples of cytochrome c oxidase (5 to 20 nmol), both phospholipid-containing and phospholipid-depleted, were mixed with 50 nmol of horse cytochrome c in a total volume of 50 to 100 ~1 and chromatographed on columns of Sephadex G-75 (0.7 x 40 cm at 22" and 0.7 x 25 cm at 4") equilibrated in solutions containing cytochrome c (0 to 20 PM) (  (35,44). The molar concentrations of cytochrome oxidase were calculated on the basrs of 2 mol of heme almol. These concentrations, corrected for the cytochrome c m the eqmhbratton buffers, were used to obtain the proportlon of cytochrome c to cytochrome oxidase.

Kinetics of Reaction
of Horse Cytochrome c with Beef Cytochrome c Onidase pH Dependence-The ascorbate-TMPD-cytochrome c oxidase activity of the Keilin-Hartree particles was studied as a function of pH in 25 mM cacodylate buffer, varying the concentration of Tris to change the pH from 5.8 to 8.4. The results ( Fig. 1) are represented in Eadie-Hofstee single reciprocal plots in which the negative reciprocal slope gives the apparent Michaelis constant of the reaction and the intercept with the abscissa gives the maximal velocity. Biphasic kinetics were observed, with an apparent K, for the initial phase of 5 x 1Oe8 M over the entire pH range examined. This value was consistently reproduced with four separate Keilin-Hartree particle preparations.
The second phase of the kinetic plots occurred with all the Keilin-Hartree particle preparations tested, but the apparent K, was not as constant as it was for the first phase, varying from 5 to 20 times that of the initial phase. In contrast to the experiments in which the cation concentration was changed, when the anion concentration was varied to change the pH in a Tris-acetate buffer, there was an mcrease in the apparent K, for both phases of the reaction as the anion concentration was decreased from 54 to 7 mM (pH 5.9 to 8.6) ( Fig. 2). At the lowest acetate concentration the kinetics became monophasic. The same results were obtained when the anion concentration was varied in a Tris-cacodylate buffer. The extrapolated V,,, of the initial phase (Fig. 2, inset) increased even more dramatically than when the cation concentration was changed (Fig. l), with an apparent inflection point at pH 7.2.
These observations suggested that anion concentration had an important influence on the kinetic behavior of the system, whereas it was less affected by the cation concentration in the ranges studied. A further examination of the effects of various anions is described below. Temperature Dependence-The apparent K, of the initial phase of the kinetics was unaffected by temperature over the range of 11 to 35". The maximal velocity of this phase doubled for each 10' increase in temperature over the same range.

Enzyme Concentration
Dependence-When the concentration of the Keilin-Hartree particles was increased over a lo-fold range there was a S-fold increase in the apparent K, for the initial phase at pH 7.8 in 25 mM Tris-cacodylate buffer. The value for this apparent K,, extrapolated to zero enzyme concentration, was 2.8 x lOma M (Fig. 3, inset). This value is remarkably close to that obtained for the dissociation constant for the binding of cytochrome c to cytochrome c-depleted mitochondria (approximately 3 x lo-* M) (45,46). As expected, the V,,, (Fig. 3, inset) was linearly dependent on the enzyme concentration over the range examined and extrapolated to zero at zero enzyme concentration.
It should be noted that in Fig. 3 higher concentrations of cytochrome c that reveal the second kinetic phase were employed with only two of the five Keilin-Hartree particle concentrations studied. One possible explanation of the increase in apparent K, with enzyme concentration is that at the higher levels of Keilin-Hartree particles a significant proportion of the cytochrome c is bound, so that the total amount of cytochrome c added no longer correctly represents the concentration of free substrate. It is not possible in this case to determine whether such depletion kinetics (47,48) actually operate since the amount of cytochrome oxidase available for reaction with externally added cytochrome c cannot be estimated with certainty for Keilin-Hartree particles. However, since disruption of the vesicles by treatment with deoxycholate (1 mg/mg of protein) according to Smith and Camerino (49) resulted in an approximately &fold increase in the V,,, of the low apparent K, phase of the reaction, as little as 12% of the enzyme may be reactive in the intact Keilin-Hartree particles, suggesting that depletion of substrate should not be kinetically important except at the highest enzyme concentrations.
However, other estimates of the amount of available oxidase are possible and are considered under "Discussion." Reaction with Purified Cytochrome c Oxidase-To determine whether the reaction pattern described above was unique to membrane-bound cytochrome c oxidase, experiments were carried out with a purified oxidase preparation. Fig. 4 shows that a kinetic behavior very similar to that observed with Keilin-Hartree particles was also obtained with the pure enzyme. At low enzyme concentrations the apparent K, is 5 x 1Om8 M, identical with that found with the membrane preparations, while at high enzyme concentrations there is an increase of K,, again suggesting the possibility of substrate depletion (see "Discussion").
Interestingly, the second kinetic phase, with the high apparent K,, was not as prominent with the pure oxidase as with the Keilin-Hartree particles (compare Figs. 1 and 4).

Effect of Anions on Cytochrome c Oxidase Activity
The monovalent anions, cacodylate, acetate, and chloride, had identical inhibitory effects. As shown in Fig. 5A, when the concentration was doubled a a-fold increase of the apparent K, of the initial phase of the kinetics was observed. However, phosphate that is present as the divalent anion at the pH of the assay (pH 7.8) had a much larger effect over the same concentration range (Fig. 5B). Furthermore, the inhibition exerted by phosphate appeared to be qualitatively different from that of the monovalent anions, being largely uncompetitive. At a concentration of 25 mM, three-quarters or more of the initial low K, phase of the kinetics had been eliminated or transformed into a high K, phase, while at 50 mM no initial phase whatsoever could be detected.
The pattern of inhibition produced by the polyvalent anions, ADP and ATP, was similar to that observed with phosphate ( Fig. 6), except that the nucleotides were effective at much lower levels, well within their physiological concentration ranges of up to 5 mM (50). Even at concentrations as low as 3 mM, ATP virtually eliminated the low apparent K, phase of the kinetics, while ADP merely caused a 33% decrease in the apparent maximal velocity of this phase.
The various affects of anions cannot be explained solely on the basis of ionic strength since the monovalent and the polyvalent anions examined exhibited qualitatively different types of inhibition.

Comparison
of Horse, Bakers' Yeast, and Euglena Cytochromes c Reactions with Cytochrome c Oxidase Examination of the reactions of four different cytochromes c with beef heart Keilin-Hartree particle oxidase revealed clear differences in the low apparent K, phase of the kinetics (Fig.  7). Both yeast cytochromes c had about double the maximal velocity of the horse protein for the low apparent K, phase. The kinetic plot for the Euglenu protein extrapolated to the same maximal velocity as for the yeast cytochromes, but gave an apparent K, value 10 times that obtained for either the horse or the yeast proteins. This overall kinetic picture (Fig. 7) may be interpreted to indicate that there are two separate enzymically active sites on the oxidase which horse cytochrome c recognizes with different affinities, as suggested by the two phases of the kinetic plot (K,, = 4 x lo-' M and K,, = 1 x lOMe M). According to such an interpretation the yeast cytochromes react with both these sites with equal high affinity (K, = 4 x 1O-8 M), while the Euglena cytochrome reacts with both with equal low affinity (4 x lo-' M).
A study of the enzyme concentration dependence of-the reaction of the yeast.cytochrome c with Keilin-Hartree particle oxidase revealed another difference from the reaction of the horse protein. Comparing Figs. 3 and 8, it is apparent that the kinetics of the yeast cytochromes c showed a more marked dependence on enzyme concentration than those for horse cytochrome c. There is strong curvature of the kinetic plot for the iso-cytochrome c of yeast at the highest enzyme concen- tration employed even though at the same and higher enzyme concentrations horse cytochrome c still gave a linear initial kinetic phase. Iso-1 cytochrome c of yeast behaved identically with the iso-cytochrome c. The curvature suggests that with the yeast proteins there is a larger degree of substrate depletion, supporting the idea that both postulated kinetic sites on the oxidase bind the yeast cytochromes c strongly, while only one of them has high affinity for the horse protein, With the pure cytochrome c oxidase the difference in activity between the horse and yeast proteins at low concentrations was even more striking (Fig. 9).
An alternative explanation for these results would be that only one kinetically active site exists on the oxidase, and that the yeast cytochromes c have twice the turnover of the horse protein at that site. However, the direct binding studies reported below, that show the presence of two cytochrome c  Binding of Cytochrome c to Purified Cytochrome c Oxidase The remarkably low apparent K, values obtained for the initial phase of the kinetics were very near the dissociation constant for cytochrome c with cytochrome c-depleted mitochondria (45,46). Direct estimates of the binding of cytochrome c to purified cytochrome c oxidase were therefore carried out to examine whether the effects of the anions and of the cytochromes c of various species on the kinetics were related to changes in binding. A tightly bound complex was formed by mixing cytochrome c and cytochrome aaS which was not dissociated by gel filtration under the conditions of the experiment. The amount of complex so formed was dependent on the concentration and type of anion present (Table I). The high affinity complex with horse cytochrome c approached a ratio of 1 molecule of cytochrome c to 1 molecule of cytochrome aaS in 25 mM Tris cacodylate, while yeast iso-cytochrome c yielded a value of about 2, and the Euglenn protein gave a ratio much smaller than 1. This is strikingly similar to the kinetic results described above that indicated the presence of two active sites on the oxidase with different affinities for cytochrome c. The same 1:l ratio was obtained with horse cytochrome c at 22 and 4" (Table I), indicating a temperature independence of the binding analogous to the temperature independence of the apparent K, of the initial phase of the kinetics. This similarity is further strengthened by the effect of phosphate, ADP, and ATP, all of which decrease the amount of tightly bound complex formed, while lower ionic strength conditions cause an increase. There was no significant difference in the binding of cytochrome c to a phospholipid-depleted as compared to a phospholipid-containing preparation of cytochrome c oxidase.
In addition to the tightly bound complexes, additional reversible binding to the oxidase preparations could be measured by the gel filtration technique (Fig. 10).   (46), or the dissociation of the product from the enzyme is a controlling factor in the reaction (52).

Reaction of Horse Cytochrome c with Keilin-Hartree Particle Succinate-Cytochrome c Reductase
When succinate-cytochrome c reductase activity was assayed polarographically (see "Experimental Procedure") under the same low ionic strength conditions employed for the ascorbate-TMPD cytochrome c oxidase activity measurements, an Eadie-Hofstee plot of the results yielded a single phase with an apparent K, (5 x lo-' M) very similar to that observed for the cytochrome c oxidase reaction (Fig. 11). Increasing cytochrome c concentrations in the range of 0.05 to 2.2 pM caused an increase in the steady state rate of oxygen consumption when succinate levels were 14 mM or higher. This result differs from the observations of Smith et al. (53) who find that at higher cytochrome c and lower succinate concentrations, a reaction preceding the reduction of cytochrome c is rate-limiting.
When succinate-cytochrome c reductase activity of Keilin-Hartree particles was measured spectrophotometrically in the presence of cyanide to inhibit cytochrome oxidase activity, the initial rates were also found to increase with the cytochrome c concentration (0.3 to 7.0 FM). However, the K, values (1 x lo-@ M to 3 x lOmB M) were higher than those obtained polarographically and were of the same magnitude as the K, values for the low affinity phase of the cytochrome c oxidase kinetics. Cyanide and ATP cause spectral changes in cytochrome oxidase (54) that might reflect the same allosteric change in the enzyme resulting in a decrease in the affinity for cytochrome c. However, a more likely explanation for the K, values obtained in the spectrophotometric reductase assay is that the large excess of cytochrome over enzyme required for this assay precludes the observation of the high affinity 1:l reaction.