The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Abstract

Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI 2 dIII 1 heterotrimer in solution, but we could find no evidence for the formation of a dI 2 dIII 2 tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotidebinding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI 2 dIII 1 heterotrimer were investigated. (a) The rate of release of NADP ؉ from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI 2 dIII 1 complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.Transhydrogenase, found in the cytoplasmic membranes of bacteria, and in the inner membranes of animal mitochondria, couples the redox reaction between NAD(H) and NADP(H) to the translocation of protons.Its function in energy metabolism, biosynthesis, and detoxification has been discussed at length (1, 2). In different organisms (and possibly in different tissues of the same organism) transhydrogenase can either utilize the proton electrochemical gradient (⌬p) to drive reduction of NADP ϩ by NADH, or it can use NADPH oxidation by NAD ϩ to augment ⌬p formation. Energy coupling in transhydrogenase is indirect. In the forward direction (Reaction 1), protein conformational changes accompanying proton translocation bring together the nicotinamide rings of the bound nucleotides to allow the redox reaction (3). This "binding-change mechanism" may share common features with energy coupling in some ion-translocating ATPases. More generally, transhydrogenase has a number of properties that make it an excellent model for understanding the principles of operation of conformationally linked pumps in biology.The polypeptide organization of transhydrogenases varies between species, but the arrangement of the three components, dI, dII, and dIII, is essentially the same in all (Fig. 1). NAD(H) binds to dI, and NADP(H) binds to dIII; these two components protrude from the membrane (into the bacterial cytoplasm or mitochondrial matrix). The dII component spans the membrane, probably in 13 or 14 transmembrane helices (reviewed in Ref. 4). There is cross-linking and hydrodynamic evidence that both the bovine (5, 6) and Escherichia coli (7) transhydrogenases have two copies each of dI, dII, and dIII...

Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

“…Further, a crystal structure of the heterotrimeric complex between R. rubrum domains I and III reveals details of the interactions in the domain I:domain III interface (14). Experiments have also focused on the transhydrogenation reaction between recombinant domains I and III in the absence of domain II (9,15,16) and shown that hydride transfer from NADH to NADP in the forward direction is fast (17,18). Trypsin sensitivity, chemical modification, and NMR experiments indicate that the formation or binding of NADPH in domain III results in significant conformational change within domain III and longer range effects in the intact enzyme (4, 19 -23).…”

“…Trypsin sensitivity, chemical modification, and NMR experiments indicate that the formation or binding of NADPH in domain III results in significant conformational change within domain III and longer range effects in the intact enzyme (4, 19 -23). The structural and kinetic data have been used to propose an "alternating site, binding change mechanism" (14,16,24) in which motion of domain III within TH is described in terms of "open" and "occluded" states. Hence, conformational change involving the soluble domain I and domain III components of TH in response to the proton motive force favors hydride transfer to NADP in the forward direction (Reaction 1), or in the reverse direction, conformational change due to NADPH binding favors outward proton translocation.…”

“…Further, a crystal structure of the heterotrimeric complex between R. rubrum domains I and III reveals details of the interactions in the domain I:domain III interface (14). Experiments have also focused on the transhydrogenation reaction between recombinant domains I and III in the absence of domain II (9,15,16) and shown that hydride transfer from NADH to NADP in the forward direction is fast (17, 18). Trypsin sensitivity, chemical…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…Isolated dI of transhydrogenase is a stable dimer [23]. Bound NAD + (or NADH depending on crystallization conditions) is readily observed in the X-ray structures of isolated dI and dI-dIII complexes [13,14,[19][20][21].…”

Fast Hydride Transfer in Proton-translocating Transhydrogenase Revealed in a Rapid Mixing Continuous Flow Device

“…21 and 22). It is a dI 2 ⅐dIII 1 heterotrimer in both the crystalline state and in solution (12,23). The two characteristic properties, (a) a capacity to catalyze a rapid redox reaction between NAD(H) and NADP(H) (or their analogues) and (b) an extremely slow rate of release of bound NADP ϩ and NADPH, suggest that the isolated complex adopts a conformation similar to that of the occluded state in the intact enzyme.…”