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Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP + . Under physiological conditions, this reaction is endergonic and must be energized by the  pmf , coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the  Escherichia coli  transhydrogenase and its reconstitution into liposomes, alone or together with the  Escherichia coli  F 1 F 0  ATP synthase. We show that  E. coli  transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H + -transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of  pmf  generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.

Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli