The crystal structure of an asymmetric complex of the two nucleotide binding components of proton-translocating transhydrogenase

Background: Membrane-bound ion translocators have important functions in biology, but their mechanisms of action are often poorly understood. Transhydrogenase, found in animal mitochondria and bacteria, links the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Linkage is achieved through changes in protein conformation at the nucleotide binding sites. The redox reaction takes place between two protein components located on the membrane surface: dr, which binds NAD(H), and dill, which binds NADP(H). A third component, dII, provides a proton channel through the membrane. Intact membrane-located transhydrogenase is probably a dimer (two copies each of dl, dII, and dill). Results: We have solved the high-resolution crystal structure of a dl:dIII complex of transhydrogenase from Rhodospirillum rubrum-the first from a transhydrogenase of any species. It is a heterotrimer, having two polypeptides of dl and one of dill. The dl polypeptides fold into a dimer. The loop on dill, which binds the nicotinamide ring of NADP(H), is inserted into the NAD(H) binding cleft of one of the dl polypeptides. The cleft of the other dr is not occupied by a corresponding dill component. Conclusions: The redox step in the transhydrogenase reaction is readily visualized; the NC4 atoms of the nicotinamide rings of the bound nucleotides are brought together to facilitate direct hydride transfer with A-B stereochemistry. The asymmetry of the dI:dIII complex suggests that in the intact enzyme there is an alternation of conformation at the catalytic sites associated with changes in nucleotide binding during proton translocation.