Interdomain hydride transfer in proton-translocating transhydrogenase

Abstract

We describe the use of the recombinant, nucleotide-binding domains (domains I and III) of transhydrogenase to study structural, functional and dynamic features of the protein that are important in hydride transfer and proton translocation. Experiments on the transient state kinetics of the reaction show that hydride transfer takes place extremely rapidly in the recombinant domain I:III complex, even in the absence of the membrane-spanning domain II. We develop the view that proton translocation through domain II is coupled to changes in the binding characteristics of NADP+ and NADPH in domain III. A mobile loop region which emanates from the surface of domain I, and which interacts with NAD+ and NADH during nucleotide binding has been studied by NMR spectroscopy and site-directed mutagenesis. An important role for the loop region in the process of hydride transfer is revealed.

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH,

“…Except for glutathione reductase (30), these enzymes follow the correlation that transfer from the pro-R (A) face of the dihydronicotinamide is favored when the glycosyl torsion angle is anti while transfer from the pro-S (B) face is favored when it is syn (23). In TH hydride transfer occurs between the A-face of NAD(H) and B-face of NADP(H) (1)(2)(3)(4). In accord with the preferred stereochemistry, the glycosyl torsion angles for NADH and NADP are anti and syn ( Figure 7), respectively.…”

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

“…They were purified as described above, and prepared for experiments as described (25). 1 H, 15 N HSQC spectra were acquired, at a protein concentration of 600 M, on a Bruker AMX 500 spectrometer, using the fast heteronuclear single quantum correlation (FHSQC) NMR spectroscopy pulse sequence (26).…”

“…Crystal structures of the dIII components of human and bovine transhydrogenases were recently described (4 -6), and the solution structure of the Rhodospirillum rubrum equivalent was determined by NMR. 1 The basic fold of dIII is similar to the classical, dinucleotide-binding domain of lactate dehydrogenase, but NADP ϩ is bound with an unusual, "reversed" orientation. The nicotinamide ring of the bound NADP ϩ is exposed on a ridge of dIII.…”

“…The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).…”

“…The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).Kinetic studies of transhydrogenase have also developed, following discoveries that fragments of the protein retain their nucleotide binding and some catalytic capacity. Thus, recombinant dI and dIII proteins, which bind their cognate nucleotides, have now been isolated from a number of species (11-16); mixtures of these proteins, even from different species, catalyze transhydrogenation reactions, albeit with properties that are modified relative to those of the complete enzyme.…”