The rate of cytochrome c2 photooxidation reflects the subcellular distribution of reaction centers in Rhodobacter sphaeroides Ga cells

Abstract

Kinetics of primary donor and cytochromes c 2 and c 1 photooxidation during continuous illumination in Rhodobacter sphaeroides Ga whole cells are nearly monophasic at low pH (<7.5) in a large domain of light intensity. After addition of MgCl 2 or at high pH (>8.5) these kinetics are clearly biphasic, whatever the excitation light intensity, demonstrating the occurrence of two compartments out of equilibrium. Light-induced difference spectra show that the ‘fast’ compartment contains cytochrome c 2 and cytochrome c 1 in equal amount, while the ‘slow’ compartment includes a large excess of cytochrome c 2. The ratio between cytochromes involved in the ‘fast’ and ‘slow’ compartments increases depending on the growth conditions (1.7 for cells grown semiaerobically, 1.2 and 0.8 for cells grown at high and low light intensities, respectively). Only the cytochrome involved in the ‘slow’ compartment is coupled to the respiratory chain. This series of results indicates that the ‘fast’ compartment corresponds to the intracytoplasmic part of the membrane, while the ‘slow’ compartment to the cytoplasmic region. For the ‘fast’ compartment the stoichiometry is independent of the growth conditions. The analysis of the slow phase of the carotenoid bandshift versus the fraction of cytochromes bc 1 complex inhibited by myxothiazol shows that the diffusion of cytochrome c 2 within the ‘fast’ compartment is restricted to domains including a single cytochromes bc 1 complex, very likely associated with two reaction centers and one cytochrome c 2 to form a supercomplex. In the ‘slow’ compartment, cytochrome c 2 is a diffusible species and the stoichiometry between reaction centers and secondary donors highly depends upon the growth conditions. At high pH or in presence of MgCl 2 the cytochrome c 2 involved in the supercomplex cannot exchange with free cytochrome c 2 in excess. Therefore, the free cytochrome c 2 can only be photooxidized by the small number of reaction centers present in the cytoplasmic part of the membrane. At low pH, charge exchange occurs via the diffusion of cytochrome c 2 between the two compartments.