STOPPED-FLOW KINETICS OF HYDRIDE TRANSFER BETWEEN NUCLEOTIDES BY RECOMBINANT DOMAINS OF PROTON-TRANSLOCATING TRANSHYDROGENASE

Transhydrogenase catalyses the transfer of reducing equivalents betwee n NAD(H) and NADP(H) coupled to proton translocation across the membra nes of bacteria and mitochondria. The protein has a tridomain structur e. Domains I and III protrude from the membrane (e.g. on the cytoplasm ic side in bacteria) and domain II spans the membrane. Domain I has th e binding site for NAD(+)/NADH, and domain III for NADP(+)/NADPH. We h ave separately purified recombinant forms of domains I and III from Rh odospirillum rubrum transhydrogenase. When the two recombinant protein s were mixed with substrates in the stopped-flow spectrophotometer, th ere was a biphasic burst of hydride transfer from NADPH to the NAD(+) analogue. acetylpyridine adenine dinucleotide (AcPdAD(+)). The burst, corresponding to a single turnover of domain III, precedes the onset o f steady state, which is limited by very slow release of product NADP( +) (k approximate to 0.03 s(-1)). Phase A of the burst (k approximate to 600 s(-1)) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k approximate to 10-50 s(-1)), which pr edominates when the concentration of domain I is less than that of dom ain III, probably results from dissociation of the domain I:III comple xes and further association and turnover of domain I. Phases A and B w ere only weakly dependent on pH, and it is therefore unlikely that eit her the hydride transfer reaction, or conformational changes accompany ing dissociation of the I:III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcP dAD(+) reduction by [4B-H-2]NADPH, and by [4B-H-1]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunne lling ro the process of hydride transfer.: Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley. R. L., Bi zouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 27 2, 27535-27538]. this suggests very close proximity of the nicotinamid e rings of the two nucleotides in the I:III complex.