Structures of the dI₂dIII₁ Complex of Proton-Translocating Transhydrogenase with Bound, Inactive Analogues of NADH and NADPH Reveal Active Site Geometries^,

Abstract

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.

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.…”

“…This takes place with the dIII component in an ''occluded state'' in which both the binding and release of NADP + and NADPH are extremely slow relative to enzyme turnover [51][52][53]. Crystal structures show that with dIII in the occluded state the pro-R hydrogen atom on C4 of the dihydronicotinamide ring of NADH (on dI) can be brought into close apposition with the si face of C4 of the nicotinamide ring of NADP + (on dIII) to effect direct, stereo-specific and rapid hydride transfer [21]. The dIII component can also adopt an ''open state'', where NADP + and NADPH can rapidly bind and dissociate, respectively, but where hydride transfer between bound NADP + and NADH is blocked [36,54].…”

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

“…This helix is of particular significance to the alternating-sites hypothesis (16,47) because, as well as making large contributions to the interaction surface between the two dI monomers, with ␣11 it also links the dI.1 and dI.2 domains of each of the monomers. Furthermore, at its N terminus is the highly conserved RQD loop which binds the (dihydro)nicotinamide ring of NAD(H), and at its C terminus is the ␤-hairpin, which is suggested to regulate changes in dI-dIII interaction during enzyme turnover (20), and see below. The energy of the interaction between the two monomers of dI.Y146A is decreased by some 30 kJ mol Ϫ1 relative to the wildtype protein.…”

“…The ␣6-helix, ␤-hairpin, and RQD loop of each dI are highlighted in more intense color. The NADP ϩ and the two molecules of H 2 NADH (tetrahydro-NADH), a close analogue of NADH, are shown in space-filling format in atom colors (20). The two Tyr 146 residues are also in atom colors in space-filling format.…”

“…Tyr 146 lies on helix ␣6, one of the two helices that link domains dI.1 and dI.2; the side chain of the residue makes H-bond contact with the carbonyl group of Phe 160 at the junction between the C terminus of ␣6 and the N terminus of the "␤-hairpin" in the symmetry-related partner polypeptide. The x-ray structures suggest that, during alternation of the sites, the ␤-hairpin shifts position and dI.1 rotates against dI.2; the pivot for rotation is in ␣6 close to Tyr 146 , and in ␣11 (20). The rotation causes a change in the conformation of the "RQD" loop, and this shifts the position of the nicotinamide ring of NADH which thus gates the hydride-transfer reaction.…”