Infrared spectroscopic evidence of a redox-dependent conformational change involving ion binding residue NqrB-D397 in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a unique respiratory enzyme that conserves energy by translocating Na(+) through the plasma membrane. Found only in prokaryotes, the enzyme serves as the point of entry of electrons into the respiratory chain in many pathogens, including Vibrio cholerae and Yersinia pestis. In this study, a combined electrochemical and Fourier transform infrared (FTIR) spectroscopic approach revealed that Na(+)-NQR undergoes significant conformational changes upon oxidoreduction, depending on the monovalent cation present (Na(+), Li(+), K(+), or Rb(+)). In the presence of the inhibitor Rb(+), additional conformational changes are evident, indicating a changed accessibility of the sodium binding sites. In electrochemically induced FTIR difference spectra, the involvement of deprotonated acid residues in the binding of cations, together with the spectral features, that point toward a monodentate binding mode for these acid residues in the oxidized form of the enzyme and bidentate binding in the reduced form could be identified. The measurements confirmed that NqrB-D397 is one of the acid residues involved in Na(+) and Li(+) binding. In the NqrB-D397E mutant, the spectral features characteristic of COO(-) groups are shifted, and a weakening of the hydrogen binding of the ion binding cluster is revealed. Finally, H-D exchange kinetics of amide protons confirmed that Na(+)-NQR adopts different conformations, with different accessibilities to the aqueous environment, depending on the cation present, which contributes to the selectivity mechanism of ion translocation.