Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

Abstract

Transhydrogenase couples the redox reaction between NADH and NADP؉ to proton translocation across a membrane. The protein has three components: dI binds NADH, dIII binds NADP ؉ , and dII spans the membrane. Transhydrogenase is a "dimer" of two dI-dII-dIII "monomers"; x-ray structures suggested that the two catalytic sites alternate during turnover. Invariant Tyr 146 in recombinant dI of Rhodospirillum rubrum transhydrogenase was substituted with Phe and Ala (proteins designated dI.Y146F and dI.Y146A, respectively). Analytical ultracentrifuge experiments and differential scanning calorimetry show that dI.Y146A more readily dissociates into monomers than wild-type dI. Analytical ultracentrifuge and Trp fluorescence experiments indicate that the dI.Y146A monomers bind NADH much more weakly than dimers. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics. However, dI.Y146A reconstituted activity in its dimeric form but not in its monomeric form, this despite monomers retaining their native fold and binding to the dI-depleted membranes. It is suggested that transhydrogenase reconstructed with monomers of dI.Y146A is catalytically compromised, at least partly as a consequence of the lowered affinity for NADH, and this results from lost interactions between the nucleotide binding site and the protein ␤-hairpin upon dissociation of the dI dimer. The importance of these interactions and their coupling to dI domain rotation in the mechanism of action of transhydrogenase is emphasized. Two peaks in the 1 H NMR spectrum of wild-type dI are broadened in dI.Y146A and are tentatively assigned to S-methyl groups of Met resonances in the ␤-hairpin, consistent with the segmental mobility of this feature in the structure.Transhydrogenase is located in the inner mitochondrial membrane of animal cells and in the cytoplasmic membrane of bacteria. It catalyzes the reduction of NADP ϩ by NADH coupled to the translocation of protons across the membrane shown in Reaction 1.The enzyme provides NADPH for biosynthesis reactions (1, 2) and for glutathione reduction in the protection of cells against oxidative stress (3-5). A role for transhydrogenase has also been implicated in glucose-stimulated insulin secretion by pancreatic ␤-cells (6).Transhydrogenase has three components. The dI component, which binds NADH, and the dIII component, which binds NADP ϩ , protrude from the membrane, and the dII component spans the membrane. Conformational changes link the redox reaction at the dI/dIII interface with proton translocation through dII (for recent reviews, see Refs. 7,8). The intact enzyme is a "dimer" of two dI-dII-dIII "monomers" (9 -12), although the disposition of dI, dII, and dIII along polypeptides is somewhat variable between species (13). Solution studies (14, 15) and x-ray structures (16 -20) reveal the profound asymmetry of a complex formed from mixtures of dI and dIII (the socalled dI 2 dIII 1 complex) of the Rhodospirillum rubrum enzyme. This was taken to suggest that transhydrogen...

Mutations In Transhydrogenase Change The Fluorescence Emission State Of Trp72 From 1La To 1Lb.

“…Mutants, I3A and M97A, were found in inclusion bodies, and were refolded and purified in a procedure developed by S.J. Whitehead, as described (18). Mutants, L339A and L339V, were also found in inclusion bodies but could not be refolded.…”

“…For thermostability determinations, the dI proteins were dialyzed against 30 mM HEPES, 10 mM (NH 4 ) 2 SO 4 , 0.5 mM dithiothreitol, pH 8.0 and their concentrations adjusted to 12.5 mM. The dependence of heat capacity on temperature was measured in a MicroCal VP-DSC, and the transition temperatures determined as described (18).…”