A catalytically active complex formed from the recombinant dI protein of Rhodospirillum rubrum transhydrogenase, and the recombinant dIII protein of the human enzyme

Transhydrogenase is a proton pump. It has three components: dI and dill pro trude from the membrane and contain the binding sites for NAD(H) and NADP(H ), respectively, and dII spans the membrane. We have expressed dIII from Ho mo sapiens transhydrogenase (hsdIII) in Escherichia coli. The purified prot ein was associated with stoichiometric amounts of NADP(H) bound to the cata lytic site. The NADP(+) and NADPH were released only slowly from the protei n, supporting the suggestion that nucleotide-binding by dIII is regulated b y the membrane-spanning dII. HsdIII formed a catalytically active complex w ith recombinant dI from Rhodospirillum rubrum (rrdI), even in the absence o f dII. The rates of forward and reverse transhydrogenation catalysed by thi s complex are probably limited by slow release from dill of NADPH and NADP( +), respectively. The hybrid complex also catalysed high rates of 'cyclic' transhydrogenation, indicating that hydride transfer, and exchange of nucle otides with dI, are rapid. Stopped-flow experiments revealed a rapid, monoe xponential, single-turnover burst of reverse transhydrogenation in pre-stea dy-state. The apparent first-order rate constant of the burst increased wit h the concentration of rrdI. A deuterium isotope effect (k(H)/k(D) approxim ate to 2 at 27 degrees C) was observed when [4B-H-1]NADPH was replaced with [4B-H-2]NADPH. The characteristics of the burst of transhydrogenation with rrdI:hsdIII differed from those previously reported for rdI:rrdIII (J.D. V enning et al., fur. J. Biochem. 257 (1998) 202-209), but the differences ar e readily explained by a greater dissociation constant of the hybrid comple x. The steady-state rate of reverse transhydrogenation by the rrdI:hsdIII c omplex was almost independent of pH, but there was a single apparent pK(a) (similar to 9.1) associated with the cyclic reaction. The reactions of the dI:dIII complex probably proceed independently of those protonation/deproto nation reactions which, in the complete enzyme, ate associated with H+ tran slocation, (C) 1999 Elsevier Science B.V. All rights reserved.