Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI 2 ⅐dIII 1 complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP ؉ is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI 2 ⅐dIII 1 complexes catalyze reduction of NADP ؉ by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s ؊1 , which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD ؉ ) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.Transhydrogenase is found in the inner membranes of animal mitochondria and in the cytoplasmic membranes of many bacteria. It couples the redox reaction between NAD(H) and NADP(H) to inward translocation of protons across the membrane (Eq. 1).The enzyme provides NADPH for biosynthesis and glutathione reduction, and in mitochondria, it also helps to control flux through the tricarboxylic acid cycle (1, 2). The relative simplicity of transhydrogenase, the emergence of methods for determining its rate of reaction in real time (3-6), and recent high resolution structural information (7-12) make it a good model for understanding the general principles of operation of conformationally coupled ion translocators. The enzyme is composed of three components. dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude into the mitochondrial matrix (or the bacterial cytoplasm), and dII spans the membrane. The intact enzyme is effectively a dimer of two dI⅐dII⅐dIII "trimers" (13,14), though there are species variations in the way the polypeptide chains are joined. The findings that the transfer of hydride-ion equivalents between the bound nucleotides on transhydrogenase is direct (3, 5) and that there is no exchange of the transferred hydride with water protons (15) together establish that coupling to proton translocation does not occur at the redox step. We proposed an NADP(H) binding change model in which NADP ϩ (or NADPH) from the solvent can only bind to (or leave from) an "open" state of the dIII component of the protein and in which the redox reaction can only take place in an "occluded" state. Association and dissociation of protons during translocation, gated by the redox state of the NADP(H), drives the protein between the open and occluded states (16, 7). Recent observations on pro...