Division of labor in transhydrogenase by alternating proton translocation and hydride transfer

Abstract

NADPH/NADP+ homeostasis is critical for countering oxidative stress in cells. Nicotinamide nucleotide transhydrogenase (TH), a membrane enzyme present in both bacteria and mitochondria, couples the proton motive force to the generation of NADPH. We present the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystallized as a symmetric dimer, with each protomer containing a putative proton channel. The holo-TH is a highly asymmetric dimer with the NADP(H)-binding domain (dIII) in two different orientations. This unusual arrangement suggests a catalytic mechanism in which the two copies of dIII alternatively function in proton translocation and hydride transfer.

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

“…Gene fusions during evolution were thought to increase the number of TM in the E. coli and mitochondrial-type enzymes [39,40]. Consistent with these views, the new X-ray structure of dII from T. thermophilus transhydrogenase (a three-subunit enzyme) has 12 TM per protomer, 3 in the a 2 -subunit, and 9 in the b subunit [22]. The most highly conserved of the transmembrane helices are TM2, TM3, TM4, TM9, TM10, and especially, TM13 and TM14 (for the TM numbering system, see Fig.…”

“…The recent structure of the membrane-spanning dII at 2.8 Å A 0 resolution, and of the holo-enzyme at 6.9 Å A 0 from Thermus thermophilus [22], provide clues as to how hydride transfer from NADH to NADP + at the interface of dI and dIII is coupled to proton translocation through dII. Distances are such that coupling must be mediated by conformational changes across the protein.…”

“…Thus, the single-subunit enzyme from animal mitochondria was thought to have 14 transmembrane helices (TM) per protomer, the Modified from Ref. [22]. The gray bars indicate the position of the membrane.…”

“…Low concentrations of Zn ++ block proton translocation through the E. coli dII channel; both FTIR [49] and X-ray absorption studies [50] implicate a His residue in the metal-ion binding but the role of bHis91 in the process is not yet clear. The conserved salt bridge between TtbD202 and TtbR254 in the hinge, which very likely has an important role in catalysis [44], is located next to the proton channel at the membrane surface [22]. Differences in electron density of the hinge region suggest different conformations in the two protomers.…”

“…Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al, 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP + /NADPH during enzyme turnover.…”