THE REDUCTION OF ACETYLPYRIDINE ADENINE-DINUCLEOTIDE BY NADH - IS IT A SIGNIFICANT REACTION OF PROTON-TRANSLOCATING TRANSHYDROGENASE, OR ANARTIFACT

Transhydrogenase is a proton pump. It has separate binding sites for N AD(+)/NADH (on domain I of the protein) and for NADP(+)/NADPH (on doma in III). Purified, detergent-dispersed transhydrogenase from Escherich ia coli catalyses the reduction of the NAD(+) analogue, acetylpyridine adenine dinucleotide (AcPdAD(+)), by NADH at a slow rate in the absen ce of added NADP(+) or NADPH. Although it is slow, this reaction is su rprizing, since transhydrogenase is generally thought to catalyse hydr ide transfer between NAD(H) - or its analogues and NADP(H) - or its an alogues, by a ternary complex mechanism. It is shown that hydride tran sfer occurs between the 4A position on the nicotinamide ring of NADH a nd the 4A position of AcPdAD(+). On the basis of the known stereospeci ficity of the enzyme, this eliminates the possibilities of transhydrog enation (a) from NADH in domain I to AcPdAD(+) wrongly located in doma in III; and (b) from NADH wrongly located in domain III to AcPdAD(+) i n domain I. In the presence of low concentrations of added NADP(+) or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD (+) by NADH. This reaction is cyclic; it ta kes place via the alternate oxidation of NADPH by AcPdAD(+) and the re duction of NADP(+) by NADH, while the NADPH and NADP(+) remain tightly bound to the enzyme. In the present work, it is shown that the rate o f the cyclic reaction and the rate of reduction of AcPdAD(+) by NADH i n the absence of added NADP(+)/NADPH, have similar dependences on pH a nd on MgSO4 concentration and that they have a similar kinetic charact er. It is therefore suggested that the reduction of AcPdAD(+) by NADH is actually a cyclic reaction operating, either with tightly bound NAD P(+)/NADPH on a small fraction(< 5%) of the enzyme, or with NAD(+)/NAD H (or AcPdAD(+)/AcPdADH) unnaturally occluded within the domain III si te. Transhydrogenase associated with membrane vesicles (chromatophores ) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD(+) b y NADH in the absence of added NADP(+)/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD(+) by NADPH). The two reactions were fully recovered upon r econstitution with recombinant domain I protein. However, after repeat ed washing of the domain I-depleted chromatophores, reverse transhydro genation activity (when assayed in the presence of domain I) was retai ned, whereas the reduction of AcPdAD(+) by NADH declined in activity. Addition of low concentrations of NADP(+) or NADPH always supported th e same high rate of the NADH --> AcPdAD(+) reaction independently of h ow often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD(+) by NADH in chromat ophores is a cyclic reaction involving nucleotides that are tightly bo und in the domain Ill site of transhydrogenase. However, in the case o f R. rubrum membranes it can be shown with some certainty that the bou nd nucleotides are NADP(+) or NADPH. The data are thus adequately expl ained without recourse to suggestions of multiple nucleotide-binding s ites on transhydrogenase.