INTERDOMAIN HYDRIDE TRANSFER IN PROTON-TRANSLOCATING TRANSHYDROGENASE

We describe the use of the recombinant, nucleotide-binding domains (do mains I and III) of transhydrogenase to study structural, functional a nd dynamic features of the protein that are important in hydride trans fer and proton translocation. Experiments on the transient state kinet ics of the reaction show that hydride transfer takes place extremely r apidly in the recombinant domain I:III complex, even in the absence of the membrane-spanning domain LT. We develop the view that proton tran slocation through domain II is coupled to changes in the binding chara cteristics of NADP(+) and NADPH in domain III. A mobile loop region wh ich emanates from the surface of domain I, and which interacts with NA D(+) and NADH during nucleotide binding has been studied by NMR spectr oscopy and site-directed mutagenesis. An important sole for the loop r egion in the process of hydride transfer is revealed. (C) 1998 Elsevie r Science B.V.