Abstract
Transhydrogenase couples proton translocation across a membrane to hydride transfer between NADH and NADP+. Previous x-ray structures of complexes of the nucleotide-binding components of transhydrogenase ("dI2dIII1" complexes) indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation. We have mutated three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase. In each mutant, turnover by the intact enzyme is strongly inhibited. Stopped-flow experiments using dI2dIII1 complexes show that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures of dI2dIII1 complexes carrying the mutations showed that their effects were restricted to the locality of the bound NAD(H). The results are consistent with the suggestion that in wild-type protein movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer.
Highlights
The transport of substances by proteins across membranes is important in many biological processes
In this study we describe the effects of mutating Arg-127, Asp-135, and Ser-138 on nucleotide binding affinity, the kinetics of hydride transfer, and the x-ray structure of the dI2dIII1 complexes
Of Mutant dI Proteins into Intact Transhydrogenase and dI2dIII1 Complexes and Measurements of Hydride Transfer Rates—Arg127 in the dI component of R. rubrum transhydrogenase was mutated to Ala and to Met; Asp-135 was mutated to Asn and Ser-138 to Ala
Summary
Transhydrogenase is driven toward the reduction of NADPϩ by NADH through the utilization of a transmembrane proton electrochemical gradient (⌬p) generated by the respiratory (or sometimes photosynthetic) electron transport chain. A switch in proton access to the translocation machinery from one side of the membrane to the other is effected by conformational changes linked to the redox state of the bound NADP(H) [8, 9]. Mixtures of separately purified dI and dIII readily form a dI2dIII1 complex (see Fig. 1), which provides a useful experimental system for investigating the conformational changes associated with the hydride transfer reaction (10 –15). The Rhodospirillum rubrum transhydrogenase has been suitable for these analyses and, has yielded high resolution structures of isolated dI and dIII and of the dI2dIII1 complex in different nucleotide-bound forms [17,18,19,20,21,22]. The x-ray structures of the dI2dIII1 complex indicate that conformational changes can occur in NAD(H) and the NAD(H)-binding pocket in the hydride transfer site [18, 20].
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