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.

Edited by Peter BrzezinskiKeywords: Transhydrogenase Membrane-protein structure Nicotinamide nucleotide Proton-pump Proton-gating a b s t r a c tThe membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP + by NADH to proton translocation. 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.

The membrane-bound nicotinamide nucleotide transhydrogenase is a key enzyme for the maintenance of metabolic balance in mammalian cells as well as in many bacteria. The enzyme resides in the mitochondrial inner membrane in eukaryotic cells or the cytoplasmic membrane in bacteria. Under normal physiological conditions, the transhydrogenase utilizes the proton motive force to drive hydride transfer from NADH to NADP+, thus generating NADPH. Among other functions, NADPH is critical for the cellular defense against reactive oxygen species. Although not the only source of NADPH, the transhydrogenase is often important, depending on cell type and physiological state. People with the most severe mutations in the Nnt gene, encoding transhydrogenase, suffer from familial glucocorticoid deficiency. Recent X-ray structures of the transhydrogenase from the hyperthermophilic bacterium Thermus thermophilus have provided key insights into how this enzyme couples proton flux across the membrane to hydride transfer. The central hypothesis from these studies focuses on the proposal that large motions of the NADP(H) binding domain (dIII), swiveling between alternating states during the catalytic cycle, are responsible for gating the proton channel in response to the redox state of bound NADP+/NADPH.

Nicotinamide nucleotide transhydrogenase (TH) is an enzyme complex in animal mitochondria and bacteria that utilizes the electrochemical proton gradient across membranes to drive the production of NADPH. The enzyme plays an important role in maintaining the redox balance of cells with implications in aging and a number of human diseases. TH exists as a homodimer with each protomer containing a proton-translocating transmembrane domain and two soluble nucleotide binding domains that mediate hydride transfer between NAD(H) and NADP(H). The three-domain architecture of TH is conserved across species but polypeptide composition differs substantially. The complex domain coupling mechanism of TH is not fully understood despite extensive biochemical and structural characterizations. Herein the progress is reviewed, focusing mainly on structural findings from 3D crystallization of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from Thermus thermophilus. A structural perspective and impeding challenges in further elucidating the mechanism of TH are discussed.

Summary The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2 Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular-dynamic simulations, we show transient water flows across a narrow pore and a hydrophobic “dry” region in the middle of the membrane channel with key residues His42α2 (chain A) being protonated and Thr214β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.