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

Abstract

Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding R1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 Å resolution in the absence of dinucleotide and at 1.9 Å resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH-vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.The energy-transducing nicotinamide nucleotide transhydrogenases of eukaryotic mitochondria and bacteria are homodimeric integral membrane proteins of monomer molecular mass of about 110 kDa. They catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD(H) 1 and the 4B position of NADP(H) in a reaction that is coupled to transmembrane proton translocation with a H + /H -stoichiometry of n ) 1 (eq 1) (1-4).In bovine submitochondrial particles, the proton motive force (pmf) accelerates the forward reaction 10-12-fold, and shifts the equilibrium toward product formation. In the reverse direction, transhydrogenation from NADPH to NAD results in outward proton translocation and creation of a pmf. Because there is essentially no difference in the reduction potential of the nicotinamide cofactors, the driving force for proton translocation coupled to the reverse reaction is the difference in binding affinities for substrates (NADPH, NAD) and products (NADH, NADP). In mammalian mitochondria, a function of TH is to produce NADPH for reduction of toxic H 2 O 2 by glutathione reductase and glutathione peroxidase.TH monomers are composed of three domains: a 400-430-residue hydrophilic domain I, a 360-400-residue hydrophobic domain II, and a 200-residue hydrophilic domain III. In mammalian TH, the ...

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

“…High-resolution structures of isolated dI [13][14][15] and dIII [16][17][18], and that of a dI-dIII complex [19][20][21], have been published during the last decade, and have afforded insights particularly into nucleotide binding and the hydride transfer step. 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.…”

“…The dIII component comprises a single, classical Rossmann fold but the NADP + (or NADPH) is bound in a non-classical manner with a reverse nucleotide orientation [16,17]. Favored binding of NADP + /NADPH relative to NAD + /NADH is ensured by interaction between the 2 0 phosphate of the AMP moiety of the former and a conserved KRS motif in loop E. The central section of this loop arches over the pyrophosphate group of the bound nucleotide forming a ''lid'' (Fig.…”

“…Studies on these and other mutants in loop E and helix D/loop D of isolated E. coli dIII reveal further consequences of interactions of the protein with NADP + and NADPH [33,34]. In crystal structures the equivalent residues to EcbD392 form hydrogen bonds with the pyrophosphate and a ribose hydroxyl group of bound NADP + /NADPH [16,17]. Its unusually high pK a , sensitive to the redox state of the bound nucleotide [35][36][37], and the fact that its substitution results in a failure in NADP + /NADPH binding [33] to isolated dIII, indicate an important catalytic role for this residue.…”

Protective Role of Methylene Blue in Alzheimer's Disease via Mitochondria and Cytochrome c Oxidase

“…The factors that trigger protein misfolding in vivo and with age are not yet clear. However, genetic polymorphism, as well as environmental and lifestyle factors may be important risk factors for Aβ peptide accumulation and aggregation [3][4][5][6].…”

“…Thus, various Aβ species containing 39-43 amino acid residues are produced [2]. The cleavage of AβPP by γ-secrtetase increases with age and in response to specific risk and genetic mutations, increasing the production of the long species of Aβ [3][4][5][6]. We will collectively refer to all the forms as Aβ.…”