Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.

G.D Eytan,E Eytan,J Rydström

doi:10.1016/s0021-9258(18)61147-0

Abstract

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.

Highlights

Purified nicotinamide-nucleotide transhydrogenase An importantaspect of the coupling mechanismisthe from beef heart mitochondria was co-reconstituted possible existence of partial reactions
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. These results suggest claimed to indicate partial reactions and the existence of a reduced enzyme intermediate [6, 7]
3-acetylpyridine adenine dinucleotide by NADH, pre- achieved by co-reconstitution of transhydrogenase and bacviously claimed to represent partial reactions, was teriorhodopsinT. hese transhydrogenase-bacteriorhodopsin found torequire NADPH

Summary

THEJOURNAOLF BIOLOGICAL CHEMISTRY

Vol 262, No 11, Issue of April 15, pp. 5015-5019,1987 Printed in U.S.A. LIGHT-DRIVENTRANSHYDROGENASE CATALYZED BY TRANSHYDROGENASEFROMBEEFHEART MITOCHONDRIARECONSTITUTEDWITHBACTERIORHODOPSIN*. 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 claimed to indicate partial reactions and the existence of a reduced enzyme intermediate [6, 7]. Even though the results indicate that this interaction is mediatebdy adelocalized rather thana localized proton-motive force, more detailed studies of this system are required before a definite conclusion can be drawn in this that transhydrogenase and bacteriorhodopsininteract regard. In this context, iwt ould be valuable to demonstratea through a delocalized proton-motive force. Reduction of thio- vesicles allow detailed studies of possible partial reactions in NADP+ byNADPH required NADH.It is concluded thetranshydrogenasereactionas well as of the coupling that these reactions do not represent partial reactions. mechanism of transhydrogenase

MATERIALS ANDMETHODS

RESULTS

DISCUSSION