Glutamate 95 in NqrE Is an Essential Residue for the Translocation of Cations in Na+-NQR

The sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) is a bacterial enzyme that oxidizes NADH, reduces ubiquinone, and translocates Na+ across the membrane. We previously identified three acidic residues in the membrane-spanning helices, near the cytosol, NqrB-D397, NqrD-D133, and NqrE-E95, as likely candidates to be involved in Na+ uptake; and replacement of any one of them by a non-acidic residue affects the Na+ dependent kinetics of the enzyme. Here, we have inquired further into the role of the NqrE-E95 residue by constructing a series of mutants in which this residue is replaced by amino acids with different charge and/or size than the glutamate of the wild-type enzyme. All of the mutants showed altered steady-state kinetics with the acceleration of turnover by Na+ greatly diminished. Selected mutants were studied by other physical methods: Membrane potential measurements showed that NqrE-E95D and A are significantly less efficient in ion transport. NqrE-E95A, Q and D were studied by transient ki...