A direct demonstration of proton translocation coupled to transhydrogenation in reconstituted vesicles.

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

“…Using TMRE and ACMA fluorescence we show that transhydrogenase establishes a pmf by translocating protons across the membrane, both when run in the forward and reverse direction. The establishment of a positive inside proton gradient in proteoliposomes during the reverse reaction has been shown before for the bovine enzyme 17 , 19 , 21 , 48 as well as for the E. coli enzyme 21 , 22 , 31 , 32 , 46 , 47 , 49 , 50 . We now show that the pmf generated during the reverse reaction is sufficient to sustain ATP synthesis by ATP synthase co-reconstituted in the same liposomes.…”

“…We show for the first time that in liposomes containing both enzymes, TH generates a sufficiently large pmf to energize ATP synthesis by the ATP synthase. While some of these experiments have been described before 17 – 20 , we are convinced that our modern straight-forward experimental setup is timely to help researchers elucidating the enigmatic molecular mechanism of energy coupled transhydrogenase.…”

“…Transhydrogenase was thus the first enzyme being demonstrated to use the energy derived from the respiratory chain without being involved in the phosphorylation machinery. Purified transhydrogenase from beef heart mitochondria was extensively studied in reconstitution experiments by Earle and Fisher 17 or Enander and Rydström 18 , including coreconstitution of transhydrogenase with ATP synthase from bovine heart 19 , 20 . Since an ATP hydrolysis generated pmf accelerated the TH forward reaction considerably, the coupling between pmf and hydride transfer was recognized to be mediated by the transport of one proton from the P - to the N -side per every hydride being transferred to form NADPH 19 – 22 .…”

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism

“…It regulates mitochondrial redox levels by coupling hydride transfer between β-nicotinamide adenine dinucleotide NAD(H) and β-nicotinamide adenine dinucleotide 2'-phosphate NADP (+) to proton translocation across the inner mitochondrial membrane. Under most physiological conditions, the enzyme produces high concentrations of NADPH, which is used for biosynthesis and oxidative stress detoxification (Earle and Fisher, 1980;Rydstrom et al, 1970;Zhang et al, 2017). NNT is regarded as a major source of NADPH and reduced glutathione in mitochondria (Ronchi et al, 2016).…”