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

Homogeneous bovine heart mitochondrial transhydrogenase was reconstituted into K+-loaded phosphatidylcholine liposomes. Transhydrogenase-catalyzed reduction of 3-acetylpyridine adenine dinucleotide by NADPH was stimulated severalfold when valinomycin was added to collapse the developing membrane potential. A rapid and extensive quenching of the fluorescence of the pH probe, 9-aminoacridine, under these conditions indicates that transhydrogenation is coupled to the acidification of the vesicle interior. A corresponding uptake of protons from the medium is demonstrated by electrode measurements that indicate 1 or less proton is translocated for each hydride ion equivalent transferred between the substrates.