Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria

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Abstract
Coupled, cytochrome-c-depleted (stripped) rat liver mitochondria reducing O2 in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in steady-state reduction of cytochrome c in excess O2 on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3-6, as obtained with reconstituted oxidase-containing vesicles. Succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase. Respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follows a Michaelis-Menten relationship with respect to O2 concentration, in accordance with the pattern found previously with the solubilized oxidase. Succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against O2 concentration, in accordance with the pattern found previously with intact uncoupled mitochondria. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate and O2 concentration during succinate oxidation under uncoupled conditions it is found that at a full reduction of cytochrome c, apparent Km for O2 is 0.9 .mu.M and maximal oxidase (aa3) turnover is 400 s-1 (pH 7.4, 30.degree. C). The redox state of cytochrome c in uncoupled systems reflects a simple steady state; redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.