cytochromes-c1 and quinone

The results are presented of an electrochemical and high-resolution spectral analysis of the heme prosthetic groups in the bc1 complex from mouse cells. To study the long-range interactions between the Qo and Qi quinone redox sites and the b heme groups, we analyzed the effects on the proximal and distal b heme groups, and the c1 heme, of inhibitors that tightly and specifically bind to the Qi or Qo redox site. A number of results emerged from these studies. (1) There is inhomogeneous broadening of the b heme alpha band absorption spectra. Furthermore, contrary to the conclusion from low-resolution spectral analysis, the higher energy transition in the split-alpha band spectrum of the bL heme is more intense than the lower energy transition. (2) Inhibitors that bind at the Qi site have significant effects upon the electronic environment of the distal bL heme. Conversely, Qo site inhibitors induced changes in the electronic environment of the distal bH heme. (3) In contrast, inhibitor binding at either site has little effect upon the midpoint potential of the distal heme. (4) Experiments in which both a Qi and a Qo inhibitor are bound at the redox sites indicate that the long-range effects of one inhibitor are not blocked by the second inhibitor; enhanced effects are often observed. (5) In the double-inhibitor titrations involving the Qo inhibitor myxothiazol, there is evidence for two electrochemically and spectrally distinct species of the bL heme group, a phenomenon not observed previously. (6) The high-resolution deconvolutions of alpha band absorption spectra allow an interpretation of these inhibitor-induced changes in terms of homogeneous broadening, inhomogeneous broadening, and changes in x-y degeneracy. The general conclusion from these experiments is that when an inhibitor binds to a quinone redox site of the cytochrome b protein, it produces local conformational changes that, in turn, are transmitted to distal regions of the protein. The ligation of the bH and bL hemes between two parallel transmembrane helices provides a mechanism by which long-distance interactions can be propagated. The lack of long-range effects upon the midpoint potentials of the heme groups suggests, however, that protein conformational changes are unlikely to be a major control mechanism for the transmembrane electron- and proton-transfer steps of the Q cycle.

Topics: Animals; Anthraquinones; Antimycin A; Benzoquinones; Binding Sites; Cell Line; Chromatography, Ion Exchange; Cytochrome b Group; Cytochromes c1; Electrochemistry; Electron Transport Complex III; Fibroblasts; Heme; Methacrylates; Mice; Oxidation-Reduction; Polyenes; Spectrophotometry; Thiazoles

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.

Topics: Antimycin A; Benzoquinones; Cytochrome b Group; Cytochrome c Group; Cytochromes c1; Dithionite; Hydroquinones; Intracellular Membranes; Kinetics; Methacrylates; Mitochondria; Models, Biological; Oxidation-Reduction; Quinones; Saccharomyces cerevisiae; Succinates; Succinic Acid; Thiazoles