Two distinct binding sites for high potential iron-sulfur protein and cytochrome c on the reaction center-bound cytochrome of Rubrivivax gelatinosus.
The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways. A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark. A spontaneous reversion of R. gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth. This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette. We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis. We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome. Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8). Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.