Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef-heart mitochondria; implications for the mechanism of proton translocation.
Transhydrogenase from beef-heart mitochondria was solubilised with Triton X-100 and purified by column chromatography. The detergent-dispersed enzyme catalysed the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH, but only in the presence of NADP+. Experiments showed that this reaction was cyclic; NADP(H), whilst remaining bound to the enzyme, was alternately reduced by NADH and oxidised by AcPdAD+. A period of incubation of the enzyme with NADPH at pH 6.0 led to inhibition of the simple transhydrogenation reaction between AcPdAD+ and NADPH. However, after such treatment, transhydrogenase acquired the ability to catalyse the (NADPH-dependent) reduction of AcPdAD+ by NADH. It is suggested that this is a similar cycle to the one described above. Evidently, the binding affinity for NADP+ increases as a consequence of the inhibition process resulting from prolonged incubation with NADPH. The pH dependences of simple and cyclic transhydrogenation reactions are described. Though more complex than those in Escherichia coli transhydrogenase, they are consistent with the view [Hutton, M., Day, J.M., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051] that, also in the mitochondrial enzyme, binding and release of NADP+ and NADPH are accompanied by binding and release of a proton. The enzyme was successfully reconstituted into liposomes by a cholate dilution procedure. The proteoliposomes catalysed cyclic NADPH-dependent reduction of AcPdAD+ by NADH only when they were tightly coupled. However, they catalysed cyclic NADP(+)-dependent reduction of AcPdAD+ by NADH only when they were uncoupled eg. by addition of carbonylcyanide-p-trifluoromethoxyphenyl hydrazone.(ABSTRACT TRUNCATED AT 250 WORDS)
Sazanov
L A
LA
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Jackson
J B
JB
D000818:Animals; D001692:Biological Transport; D002384:Catalysis; D002417:Cattle; D003902:Detergents; D006859:Hydrogen / Q000378:metabolism*; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D011522:Protons; D012995:Solubility; D013367:Submitochondrial Particles / Q000201:enzymology; D013696:Temperature
D003902:Detergents; D008081:Liposomes; D011522:Protons; D006859:Hydrogen; D009250:NADP Transhydrogenases