STUDIES ON RECONSTITUTION OF THE RHODOSPIRILLUM-RUBRUM NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE

The energy-transducing nicotinamide nucleotide transhydrogenase of Rho dospirillum is composed of 3 subunits alpha 1, alpha 2 and beta, with M(r) values, respectively, of 40.3, 14.9 and 47.8 kDa. Subunit alpha 1 is water-soluble, loosely bound to chromatophores, and can be easily and reversibly removed. Subunits alpha 2 and beta are integral membran e proteins, and their removal from chromatophores requires the use of detergents. Treatment of chromatophores with various detergents inhibi ted reconstitution of transhydrogenase activity when al was added to t he detergent-treated chromatophores. This apparent inhibition could be reversed by addition of a divalent metal ion. The best condition for extraction of alpha 2/beta from chromatophores was the use of 1% deoxy cholate in the presence of 0.34 M KCI. Under these conditions, the ext racted alpha 2/beta mixed with purified al was completely inactive, bu t gained full activity when the assay medium was supplemented with 2-3 mM MgCl2 or CaCl2. It was shown that metal ions had little effect on the apparent K-m of substrates, but greatly increased the affinity bet ween purified alpha 1 and the detergent-treated or detergent-solubiliz ed alpha 2/beta. It seems possible that the R. rubrum transhydrogenase contains a detergent-extractable metal ion, which is required for pro per binding of the soluble alpha 1 subunit to the chromatophore-bound alpha 2/beta subunits.