The site of inhibition by 5,5′-dithiobis(2-nitrobenzoate) in ubiquinol:Cytochrome c oxidoreductase

In 5,5′-dithiobis(2-nitrobenzoate) (DTNB)-treated succinate:cytochrome c reductase, the electron transfer from duroquinol to cytochrome c is inhibited due to the fact that the Rieske Fe-S cluster and, consequently, cytochrome c, are no longer reducible by substrate. The finding that, after this treatment, cytochrome b is still reducible by substrate in the absence of antimycin, but not in its presence, is consistent with a Q-cycle mechanism for the electron transfer through QH₂:cytochrome c oxidoreductase. The inhibitory effect of DTNB and its effect on the EPR spectrum of the [2Fe-2S] cluster suggest that it prevents either the binding of ubiquinone in the vicinity of this cluster or the interaction between the Fe-S protein and a ubiquinone-binding protein.     

Kinetics of flash-induced electron transfer between bacterial reaction centres,mitochondrial ubiquinol: Cytochrome c oxidoreductase and cytochrome c

Ascorbate-reduced horse heart cytochrome c reduces photo-oxidized bacterial reaction centres with a second-order rate constant of (5–8) · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. In the absence of cytochrome c, the cytochrome c₁ in the ubiquinol:cytochrome c oxidoreductase is oxidized relatively slowly (k = 3.3 · 10⁵ M^?1 · s^?1). Ferrocytochrome c binds specifically to ascorbate-reduced reductase, with a K_d of 0.6 μM, and only the free cytochrome c molecules are involved in the rapid reduction of photo-oxidized reaction centres. The electron transfer between ferricytochrome c and ferrocytochrome c₁ of the reductase is rapid, with a second-order rate constant of 2.1 · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. The rate of electron transfer from the Rieske iron-sulphur cluster to cytochrome c₁ is even more rapid. The cytochrome b of the ubiquinol:cytochrome c oxidoreductase can be reduced by electrons from the reaction centres through two pathways: one is sensitive to antimycin and the other to myxothiazol. The amount of cytochrome b reduced in the absence of antimycin is dependent on the redox potential of the system, but in no case tested did it exceed 25% of the amount of photo-oxidized reaction centres.     

A myxothiazol-sensitive Q-binding protein isolated from Chromatium vinosum

A quinol: ferricytochrome c oxidoreductase has been isolated from chromatophores of Chromatium vinosum by two procedures, involving extraction by bile salts and methanol, respectively. The steady-state kinetics indicate a random mechanism, with a K_m for 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol of 1.1 μM and for the acceptor cytochrome c 1.75 μM. The enzyme is inhibited by myxothiazol, competitively with respect to quinol, with a K_i of about 2.3 μM. The protein reacts with ubiquinol produced by the succinate: Q oxidoreductase in submitochondrial particles or isolated succinate: cytochrome c reductase and can partially restore activity to myxothiazol-inhibited, antimycin-sensitive ubiquinol: cytochrome c oxidoreductase. The protein is considered to be analogous to the postulated myxothiazol-sensitive Q-binding protein in ubiquinol: cytochrome c oxidoreductase.