Reducibility of cytochromeb in mitochondrial inner membrane

Abstract

When mitochondrial inner membrane was disintegrated into Complex I-III, IV, and oligomycin-sensitive ATPase, about 50% of cytochromeb in Complex I-III was readily reduced with NADH, as judged by the appearance of a peak at 562 nm, while in whole mitochondria less than 25% of cytochromeb was reduced by succinate. On addition of antimycin to the substrate-reduced Complex I-III, cytochromeb was further reduced to 71% of the total, and the peak at 562 nm was red-shifted to 564 nm as in the case of dithionite reduction. These results indicate that the 562 nm and 564 nm peaks, at 29°C correspond, respectively, tob ₅₆₀ andb _562.5 at 77°K of Davis et al. [7] and to “b _K” and “b _T” of Chance et al. [2]. When Complex I-III and oligomycin-sensitive ATPase were reconstituted to form a membrane, about 60% of cytochromeb in Complex I-III was readily reduced with NADH. In this case the 562 nm peak was not red-shifted. However, the difference spectrum of NADH-reduced membraneminus that in the presence of deoxycholate showed a peak at 565 nm. A mirror image of the difference spectrum was obtained on addition of an uncoupler,m-chlorocarbonyl cyanide phenylhydrazone. This is characteristic for “b _T”. These results support the idea that the occurrence of spectral peaks of “b _T” and “b _K” is not due to two species but to single species