Bioenergetic factors controlling in vitro phosphorylation of LHI alpha (B870) polypeptides in membranes isolated from Rhodobacter capsulatus

Membranes of Rhodobacter capsulatus strain U43 (pTX35) showed qualitatively very similar phosphorylation patterns under in vitro and in vivo conditions. In vitro, it was irrelevant whether the phosphate source was orthophosphate or ATP. Inhibitors of electron transport did not inhibit light-harvesting complex I (LHI alpha) (B870) polypeptide phosphorylation, except for o-phenanthroline, which was strongly inhibitory. Redox conditions regulated the amount of protein phosphorylated; external redox potentials between +200 and +300 mV promoted the reaction. Phosphorylation was inhibited by uncouplers such as carbonyl cyanide nz-chlorophenyl hydrazone and nigericin plus valinomycin plus potassium ions. Inhibitors of the H+-ATPase were also inhibitory when the phosphate source was [P-32]P-i or [gamma-P-32]ATP. From these results, it was concluded that an operative reaction center, a coupled membrane, and external redox potentials higher than +200 mV are required for optimum LHI alpha phosphorylation. We also demonstrated that phosphorylation of LHI alpha polypeptide occurs before insertion into the membrane and that phosphate is preferentially incorporated into specific domains within the cytoplasmic membrane. Intracytoplasmic membranes, identified here as light membranes, were found to contain a dephosphorylated LHI alpha polypeptide.