Proton-translocating Nicotinamide Nucleotide Transhydrogenase

Abstract

The nicotinamide nucleotide transhydrogenase of bovine mitochondria is a homodimer of monomer M(r) = 109,065. The monomer is composed of three domains, an NH2-terminal 430-residue-long hydrophilic domain I that binds NAD(H), a central 400-residue-long hydrophobic domain II that is largely membrane intercalated and carries the enzyme's proton channel, and a COOH-terminal 200-residue-long hydrophilic domain III that binds NADP(H). Domains I and III protrude into the mitochondrial matrix, where they presumably come together to form the enzyme's catalytic site. The two-subunit transhydrogenase of Escherichia coli and the three-subunit transhydrogenase of Rhodospirillum rubrum have each the same overall tridomain hydropathy profile as the bovine enzyme. Domain I of the R. rubrum enzyme (the alpha 1 subunit) is water soluble and easily removed from the chromatophore membranes. We have isolated domain I of the bovine transhydrogenase after controlled trypsinolysis of the purified enzyme and have expressed in E. coli and purified therefrom domain III of this enzyme. This paper shows that an active bidomain transhydrogenase lacking domain II can be reconstituted by the combination of purified bovine domains I plus III or R. rubrum domain I plus bovine domain III.

Catalytic Properties of Hybrid Complexes of the NAD(H)-Binding and NADP(H)-Binding Domains of the Proton-Translocating Transhydrogenases from Escherichia coli and Rhodospirillum rubrum

“…Although some domain shuffling is observed (26), the tripartite arrangement has persisted in all known enzymes. The observations described here and elsewhere (14), that hybrid mixtures of the two peripheral subunits/domains of transhydrogenases from R. rubrum, E. coli, and B. taurus are catalytically competent, further indicate that the formation of active complexes is highly tolerant of substantial changes in the primary sequences. This is all the more remarkable in view of the fact that hydride transfer between nucleotides is direct (16).…”

“…Nondenaturing size-exclusion chromatography showed that, in accordance with previous reports (9, 13), both ecI and rrI were isolated as dimers. The ecIII and rrIII behave anomalously on nondenaturing size-exclusion chromatography (9,13,14). Both the ecIII and rrIII preparations used in the present study contained approximately 20% bound NADPH and 80% bound NADP + , as judged by optical absorption analysis for ecIII (9) and by nucleotide extraction procedures for rrIII (13,23).…”

“…On the basis of kinetic experiments, there is an emerging view that proton translocation is conformationally coupled to events at the level of NADP(H) binding and release (7)(8)(9). dI proteins from E. coli (9,10) and from R. rubrum (11), and dIII from E. coli (9), R. rubrum (12,13), and Bos taurus (12,14), have recently been overexpressed in water-soluble form, purified, and partially characterized. The dI proteins are isolated as apoprotein dimers, which bind NADH with moderately high affinity and NAD + with lower affinity (15).…”

“…Yamaguchi and Hatefi (14) showed that a hybrid mixture of domain I from R. rubrum transhydrogenase and domain III from the bovine enzyme was capable of catalyzing transhydrogenation. In this report, other hybrid systems are explored, using domains from R. rubrum and E. coli transhydrogenases.…”

Site-Directed Mutagenesis of Charged and Potentially Proton-Carrying Residues in the β Subunit of the Proton-Translocating Nicotinamide Nucleotide Transhydrogenase from Escherichia coli. Characterization of the βH91, βD392, and βK424 Mutants

“…Recently, the genes of the soluble AA subunit of R. rubrum transhydrogenase (5), domain I of E. coli (6,7), and domain III of E. coli (7), bovine (8, see also 9), and R. rubrum (8,10) have been overexpressed and the gene products isolated and characterized. Interestingly, with the exception of the E. coli preparation, the combination of the expressed domains I and III, i.e., in the absence of the proton-conducting domain II, results in low forward/reverse transhydrogenase activities, but a highly active so-called cyclic reduction of AcPyAD + by NADH mediated by bound NADP(H) (7)(8)(9)(10). In contrast to domain I and the wild-type enzyme, all domain III preparations contained the expected dinucleotide, i.e., NADP(H), suggesting a pronounced regulation of the NADP(H) site by domain II.…”