Flash-induced electron transfer through mitochondrial QH 2:cytochrome c oxidoreductase in the presence of bacterial reaction centres and cytochrome c. Analysis of subsequent processes and effect of inhibitors

Abstract

In a system containing reaction centres isolated from Rhodopseudomonas sphaeroides mutant R26, and variable amounts of horse-heart cytochrome c and bovine-heart mitochondrial QH 2:cytochrome c oxidoreductase in a medium containing 2 mM ascorbate and 0.1 μM phenazine methosulphate, electron transfer was induced by a single flash. Three distinct phases of electron transfer can be distinguished: the first event is the oxidation of cytochrome c, and this is followed by an equilibration between cytochrome c, cytochrome c 1 and the Rieske [2Fe-2S] cluster. The actual rates of these processes depend on the concentrations of cytochrome c and the reductase. The slower third phase is the oxidation of ubiquinol, which can follow two pathways: one sensitive to antimycin and one sensitive to myxothiazole. The antimycin-sensitive pathway ( t 1 2 ≈ 10 ms ) is an equilibration between the Q/QH 2 couple and cytochrome b, but may also include a direct reduction of cytochrome b by the Q B of the reaction centres. The myxothiazole-sensitive pathway is a coupled reduction of cytochrome b and the Rieske [2Fe-2S] cluster which rapidly equilibrates with cytochromes c 1 and c. Both pathways are sensitive to 7-( n-heptadecyl)mercapto-6-hydroxy-5,8-quinoline quinone, but with different affinities. In the absence of inhibitors the initial reduction of cytochrome b (via both pathways) is followed by a net oxidation which is the resultant of a continuing reduction (together with the reduction of the Rieske [2Fe-2S] cluster) and an oxidation (via the antimycin-sensitive site) by quinone. The results are discussed in the light of linear and cyclic models proposed to explain electron transfer between cytochromes b and c. It is concluded that only the Q-cycle model fits the present experimental data.