Transhydrogenase is located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria. It couples the transfer of reducing equivalents of NAD(H) and NADP(H) to proton pumping. The protein has a tripartite structure and its dII component spans the membrane. The dI and dIII components protrude outside the membrane and have binding sites for NAD+/NADH and NADP+/NADPH respectively. Proton pumping is probably coupled to changes in the binding affinities of dIII for NADP+ and NADPH.

Crystals have been obtained for the NADP(H)-binding component, dIII of human-heart transhydrogenase. Diffraction data were collected on a single cryo-cooled Se-Met crystal on beamline ID14/4 to a resolution of 2.5 Å at three wavelengths corresponding to the inflexion point, peak and remote wavelength of the Se K-edge. Initially a 55° wedge of data was collected at each wavelength, followed by a second 40° wedge (space group P4122 with a = 58.1 Å and c = 250.8 Å). Each diffraction image on the ADSC Quantum 4 detector was a 1.0° oscillation with a 1 second exposure; the time to collect the complete data set was 62 minutes. The resolution was then extended to 1.9 Å using a second crystal. The program SOLVE [1] was used to locate 14 out of a possible 18 Se positions and subsequent phase determination and solvent flattening allowed the fitting of the polypeptide chain, followed by refinement of the structure.

The structure of the dIII component resembles the classical "Rossman" fold, Figure 14 [2]. However, NADP+ binds to dIII with a reversed orientation. There is a Gly-X-Gly_X-X-Ala/Val fingerprint in the first babab motif of dIII, but it has a different function to the classical Rossman structure. The nicotinamide ring of NADP+ is located on a ridge where it is exposed to interaction with NADH on the dI subunit. Two distinctive features of the dIII structure are the helix-D/loop-D which projects from the b-sheet and loop-E which forms a lid over the bound NADP+.

Thus the helix-D/loop-D interacts with the bound nucleotide, with loop-E and probably the membrane spanning second domain. Changes in the ionisation and conformation of the helix-D/loop-D resulting from proton translocation through dII may be responsible for the changes in affinity of dIII for NADP+ and NADPH, that drive the reaction.

[1] T.C.Terwilliger, J. Berendzen, Acta Cryst., D55, 849-861 (1999).
[2] S.A. White, S.J. Peak, S. McSweeney, G. Leonard, N.P.J. Cotton, J.B. Jackson, Structure, 8, 1-12 (2000).

S.A. White (a), S.J. Peak (a), S. McSweeney (b), G. Leonard (c), N.P.J. Cotton (a), J.B. Jackson (a).

(a) School of Biosciences, University of Birmingham (UK)
(b) EMBL Grenoble Outstation (France)
(c) ESRF