CYCLIC-REACTIONS CATALYZED 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 Tri ton X-100 and purified by column chromatography. The detergent-dispers ed enzyme catalysed the reduction of acetylpyridine adenine dinucleoti de (AcPdAD(+)) by NADH, but only in the presence of NADP(+). Experimen ts showed that this reaction was cyclic; NADP(H), whilst remaining bou nd to the enzyme, was alternately reduced by NADH and oxidised by AcPd AD(+). A period of incubation of the enzyme with NADPH at pH 6.0 led t o 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 descr ibed above. Evidently, the binding affinity for NADP(+) increases as a consequence of the inhibition process resulting from prolonged incuba tion with NADPH. The pH dependences of simple and cyclic transhydrogen ation reactions are described. Though more complex than those in Esche richia coli transhydrogenase, they are consistent with the view [Hutto n, M., Day, J.M., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Bioche m. 219, 1041-1051] that, also in the mitochondrial enzyme, binding and release of NADP(+) and NADPH are accompanied by binding and release o f a proton. The enzyme was successfully reconstituted into liposomes b y a cholate dilution procedure. The proteoliposomes catalysed cyclic N ADPH-dependent reduction of AcPdAD+ by NADH only when they were tightl y coupled. However, they catalysed cyclic NADP(+)-dependent reduction of AcPdA(+) by NADH only when they were uncoupled eg. by addition of c arbonylcyanide-p-trifluoromethoxyphenyl hydrazone. These observations are evidence that the proton binding and release which accompany NADP( +) binding and release, respectively, take place on the inside of the vesicle, and that they are components of the electrogenic processes of the enzyme.