Abstract
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.
Highlights
Kinetics of Reaction of Horse Cytochrome chrome c Onidase c with Beef CytopH 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 second phase of the kinetic plots occurred with all the Keilin-Hartree particle preparations tested, but the apparent
The present experiments indicate that this low apparent K, phase, together with the commonly studied higher K, phase of the kinetics of cytochrome c oxidation, are related to the two binding sites of different affinity that were found for cytochrome c on purified cytochrome c oxidase
Summary
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. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. 4. At low concentrations bakers’ yeast iso-1, bakers’ yeast iso-2, horse, and Euglena cytochromes c exhibited very different activities and kinetic patterns in their reactions with cytochrome c oxidase, while at high concentrations all attained. The different proportions of these cytochromes c correlated with the amounts of low apparent of high affinity complex formed with purified cytochrome. The apparent K, for cytochrome c oxidase. c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 1Om8 M), suggesting the came site serves both reactions
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