Dynamic Control on the Rate of the Reduction of the b Type Cytochromes in Submitochondrial Particles

Abstract

1. In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first-order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of chtochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall-off in the rate of cytochrome b reduction. 2. The biphasic reduction of cytochrome b was observed over a wide temperature range (0--30 degrees C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat-heart mitochondria. With rat-heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone or oligomycin. 3. In both the rapid and the slow phases, the rate of reduction of cytochrome b-561 was equal to that of b-565. Thus both cytochromes b-561 and b-565 were affected by the mechanism which determined the reduction-rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. 4. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by teh experimental conditions. When antimycin was used as the only inhibitor, 96% of the b-type cytochromes were reduced in the rapid phase. If the c and a-type cytochromes were first reduced by ascorbate and tetramethyl-p-phenylenediamine in the presence of KCN and antimycin, all the b-type cytochromes were fully reduced at the slow-rate. 5. With succinate, the rate of the rapid phase depended on the activation level of the succinic-dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). 6. It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone. 7. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.