Characterization of the Component, which Controls the Transformation between the Kinetic Forms of the b Cytochromes

Michael EISENBACH, Menachem GUTMAN

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Abstract

In the presence of KCN and a saturating concentration of antimycin the reduction of the b‐type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:bA– the active form which was reduced in the rapid phase, and bs– the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, named “Y”, located between cytochromes b and c1. Pre‐treatment with ascorbate plus N,N,N1,N1‐tetramethyl‐p‐phenylenediamine transforms all the b‐type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name “dynamic control mechanism” was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107–116]. Increasing concentrations of antimycin (0–2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of “Y” and thus permits the observation of the biphasic phenomenon, but that it is not essential for the operation of this dynamic control mechanism. Substituting antimycin with 2‐heptyl‐4‐hydroxyquinoline N‐oxide (HpHOQnO) as an inhibitor, did not change significantly the pattern of the kinetics; i.e. the reduction was still biphasic. Omission of KCN from the reaction mixture or pre‐treatment with ascorbate plus tetramethylphenylenediamine did not affect the kinetics of the reduction (in contrast to the experiments with antimycin as inhibitor). From these phenomena it is concluded that the inhibition sites of antimycin and HpHOQnO are not identical. Antimycin is located on the substrate side of “Y” and HpHOQnO is located on its oxygen side. This assumption is supported by measuring the effect of an oxidant, added in the presence of antimycin or HpHOQnO, on the rate of the reduction of cytochrome b. After aerobic reduction of submitochondrial particles by succinate in the presence of KCN, either antimycin or HpHOQnO was added, followed by ferricyanide. With antimycin, a rapid reduction was observed after the addition of the oxidant, followed by a slow reaction. In the presence of HpHOQnO, the addition of ferricyanide did not accelerate the reduction. On the contrary it induced a rapid oxidation of cytochrome b followed by a slow monophasic reduction. If the order of the additions was reversed, i.e. first K3Fe(CN)6 and then either antimycin or HpHOQnO, a biphasic reduction was observed in either case. It is suggested, that a redox equilibrium exists between “Y” and cytochrome c1, and that this equilibrium is sensitive to HpHOQnO but not to antimycin.

Original languageEnglish
Pages (from-to)223-230
Number of pages8
JournalEuropean Journal of Biochemistry
Volume59
Issue number1
DOIs
StatePublished - Nov 1975

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