Bovine heart mitochondrial transhydrogenase catalyzes an exchange reaction between NADH and NAD+.
Reconstitution of homogeneous bovine heart mitochondrial transhydrogenase into phosphatidylcholine liposomes results in a greater than 80% inhibition of NADPH leads to 3-acetylpyridine adenine dinucleotide (AcPyAD+) transhydrogenation. This coupled rate is stimulated 5-fold by addition of protonophore to the rate observed with unreconstituted enzyme. In the absence of uncoupler, addition of low concentrations (10 microM) of NADH promotes by about 3-fold the rate of reduction of AcPyAD+ in the presence of NADPH. In the absence of NADPH, no reduction of AcPyAD+ by NADH occurs. Addition of valinomycin to K+-loaded proteoliposomes stimulated NADPH leads to AcPyAD+ transhydrogenation to the uncoupled rate and allowed the uptake of protons from the medium. In the absence of valinomycin, the rapid reduction of AcPyAD+ seen on addition of NADH was not coupled to proton translocation. In the presence of valinomycin, NADH addition neither equilibrated protons across the liposomal membrane nor affected the stoichiometry of proton uptake to hydride ion transfer (H+:H- ratio). Addition of NADH to proteoliposomes in the presence of AcPyAD+ and NADPH resulted in an initial rapid formation of reduced 3-acetylpyridine adenine dinucleotide comparable to the amount of NADH added. Thereafter, the rate of AcPyAD+ reduction returned to that seen in the absence of NADH. These results suggest that the NADH requirement for AcPyAD+ reduction is stoichiometric rather than catalytic. Stereospecificity studies demonstrate that reconstituted transhydrogenase catalyzes a direct transfer of a hydride ion equivalent from the 4A locus of NADH to the 4A locus of the NADH product in the presence, but not in the absence, of NADPH. It is proposed that NADH leads to NAD+ transhydrogenation represents a partial reaction of NADPH leads to NAD+ transhydrogenation which involves the participation of a reduced-enzyme intermediate.
D000818:Animals; D002417:Cattle; D003067:Coenzymes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D014316:Tritium
D003067:Coenzymes; D009243:NAD; D014316:Tritium; C005941:3-acetylpyridine adenine dinucleotide; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases