Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotidetranshydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants

Conserved and semiconserved acidic and basic residues of the beta subunit o f the proton-pumping nicotinamide nucleotide transhydrogenase from Escheric hia coli potentially involved in proton pumping were investigated. Out of 1 6 charged residues studied, 6 have not been previously investigated. The mo st dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP(+) by NADH (forward reaction) and reduction of 3-ace tylpyridine-NAD(+) by NADPH (reverse reaction) to lower pH. This mutant cat alyzed a cyclic reduction of 3-acetylpyridine-NAD(+) by NADH in the presenc e of NADP(H) with a pH profile also shifted toward a lower pH. These. resul ts are consistent with a mechanism where the normal forward and reverse rea ctions are indeed limited by protonation/deprotonation of beta H91. The cyc lic reaction was affected by mutations of beta H91, probably through confor mational changes involving the active NADP(H) site. The beta D392A mutant w as inactive with regard to forward and reverse reactions, but showed a wild -type-like pH dependence: for the partly active cyclic reaction. However, K -m,K-app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydroge nases contain a conserved beta K424-beta R425-beta S426 sequence that has b een proposed to be important for NADP(H) binding. beta K424R was strongly i nhibited and showed an 18-fold increased K-m,K-app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected al l NADP(H)-linked activities and essentially abolished the unspecific intera ction of NAD(II) with this site. The pH dependences of the forward and reve rse reactions, as well as the cyclic reaction, were shifted to a lower pH a s compared to the wild-type enzyme, and the salt dependence was also altere d.