Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped‐flow spectrophotometer, there 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 of steady state, which is limited by very slow release of product NADP+ (k≈0.03 s−1). Phase A of the burst (k≈600 s−1) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k≈10−50 s−1), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I : III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I :III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B‐2H]NADPH, and by [4B‐1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27 535−27 538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I :III complex.