Changes in oxidation-reduction potential of cytochrome b observed in the presence of antimycin A

The cytochrome b of sonic particles of mitochondria or the isolated segment of the respiratory chain containing cytochromes b and c 1 (Complex III) was 80–95% reducible with Q 1H 2 (ubiquinol-5) in the presence of antimycin plus selected electron acceptors added externally (i.e., oxidants which reacted preferentially with respiratory components on the oxygen side of the point of inhibition by antimycin) such as oxygen or ferricyanide depending on whether sonic particles or isolated Complex III was used. In contrast, less than 40% of the cytochrome b was reduced by Q 1H 2 in the absence of either antimycin or the external electron acceptor. In the presence of antimycin ascorbate or mercaptoethanol, which behaved as mild reducing agents, completely inhibited the reduction of cytochrome b by Q 1H 2. Reduction of cytochrome b by varying mixtures of Q 1 and Q 1H 2 yielded data which could be fitted by potentiometric titration curves. Midpoint potentials of cytochrome b estimated from these curves obtained in the presence of antimycin and added electron acceptor gave values of E m7 = 0.15–0.19 V; whereas, E m7 values of ca. 0.05 V were obtained in the same system but without the added electron acceptor. In the absence of both antimycin and electron acceptor, titration curves corresponding to a cytochrome b with low values of E m at low potentials ( E h) but with anomalously steep slopes were obtained. The ΔE m ΔpH between pH values 6.0 and 7.0 for cytochrome b in the presence of antimycin and an electron acceptor was −0.05 V at 0 ° which corresponds to the dissociation of one proton from cytochrome b concomitant with oxidation of cytochrome b. The requirement of both antimycin and an added electron acceptor for measurement of the high potential form of cytochrome b is strongly suggestive that the state of oxidation of a component distinct from cytochrome b controls the midpoint potential of cytochrome b. Only when this component is in its oxidized form is cytochrome b in its high-potential state.