Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.
Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001436:Bacteriorhodopsins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D008027:Light; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000378:metabolism*
D009243:NAD; C048483:3-acetylpyridine-adenine dinucleotide phosphate; D009249:NADP; D001436:Bacteriorhodopsins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases