Characterization of Mutants of βHistidine91, βAspartate213, and βAsparagine222, Possible Components of the Energy Transduction Pathway of the Proton-Translocating Pyridine Nucleotide Transhydrogenase of Escherichia coli

The roles of three residues (βHis91, βAsp213, and βAsn222) implicated in energy transduction in the membrane-spanning domain II of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli have been examined using site-directed mutagenesis. All mutations affected transhydrogenation and proton pumping activities, although to various extents. Replacing βHis91 or βAsn222 of domain II by the basic residues lysine or arginine resulted in occlusion of NADP(H) at the NADP(H)-binding site of domain III. This was not seen with βD213K or βD213R mutants. It is suggested that βHis91 and βAsn222 interact with βAsp392, a residue probably involved in initiating conformational changes at the NADP(H)-binding site in the normal catalytic cycle of the enzyme (M. Jeeves et al. (2000) Biochim. Biophys. Acta 1459, 248–257). The introduced positive charges in the βHis91 and βAsn222 mutants might stabilize the carboxyl group of βAsp392 in its anionic form, thus locking the NADP(H)-binding site in the occluded conformation. In comparison with the nonmutant enzyme, and those of mutants of βAsp213, most mutant enzymes at βHis91 and βAsn222 bound NADP(H) more slowly at the NADP(H)-binding site. This is consistent with the effect of these two residues on the binding site. We could not demonstrate by mutation or crosslinking or through the formation of eximers with pyrene maleimide that βHis91 and βAsn222 were in proximity in domain II.