Oxygen dependent electron transfer in the cytochrome bc1 complex and the interaction between Rhodobacter sphaeroides bc1 complex and cytochrome c or c2

Abstract

Scope and Method of Study:

Although it has been well recognized that the bc1 complex also generates a small amount of superoxide anion during the catalytic reaction, the involvement of molecular oxygen in the regulation of the bc1 electron transfer reaction was never noticed until now. The mechanism of interaction between cytochromes c1 in bc1 complex and c (or c2 in bacterial system) has been extensively studied but not been concluded. To elucidate the role of oxygen in the electron transfer catalyzed by cytochrome bc1 complex and superoxide generation, we assayed the electron transfer activity of bc1 complexes, measured pre-steady b and c1 reduction in wild type of Rhodobacter sphaeroides cytochrome bc1, bL or bH lacking mutants by ubiquino as well as superoxide generation under aerobic and anaerobic conditions. To clarify nature of interaction between cytochrome c1 and c or c2, we examined the interaction under ionic strength conditions by using gel filtration, co-precipitation, fast kinetics and DSC. We also constructed mutant cytochrome c1 water soluble head domain to test the cytochrome c1 and c or c2 interaction under different ionic strength conditions.

Findings and Conclusions:

Our results demonstrated that oxygen is involved in the regulation of the electron transfer catalyzed by cytochrome bc1 complex. Oxygen increases the electron transfer activity of the bc1 complex up to 50%, depending on the intactness of the complex. Oxygen enhances the pre-steady reduction rate of cytochrome bL and has no effect on the reduction of cytochrome bH. By using a heme bL or bH knockout R. sphaeroides bc1 mutant complex, the site affected by oxygen in the electron transfer sequence was identified to be cytochrome bL. The interaction between Rhodobacter sphaeroides cytochrome bc1 and c or c2 is electrostatic. A net oxidation of cytochrome c1 and c was observed after mixing reduced c1 and oxidized c together under low ionic strength condition. The effect of ionic strength on the interaction between bc1 and c is more than the interaction between bc1 and c2.