The Role of Acid Residues in Na+ Uptake and Binding in Na+-NQR from Vibrio Cholerae

Abstract

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the gateway for electrons into the respiratory chain of Vibrio cholerae and many other pathogenic bacteria. Na+-NQR is unique among respiratory enzymes in that it pumps sodium rather than protons.The character of Na+ binding to the enzyme is crucial to understanding the coupling between Na+ translocation and the redox reaction. We are using functional studies_steady state and transient kinetics and equilibrium binding_in combination with site-directed mutagenesis to investigate the interaction of Na+ with Na+-NQR. Recently, we have focused on conserved acid residues in membrane spanning regions as candidates for Na+ binding site ligands. Here, we describe results on two of these residues, which are both involved in Na+ uptake by the enzyme: NqrB-D397 and NqrE-E95.Replacement of either residue by a neutral amino acid (Ala) results in a large increase in the apparent Km for Na+. In the case of NqrB-D397, replacement by Glu or Cys, produced smaller changes in Kmapp, indicating that the size and charge of the residue at this position both modulate Na+ binding. Stopped-flow kinetic measurements show that mutations at both positions exert their effect specifically at one internal electron transfer step: 2Fe-2S center aFMNC. These results are consistent with the earlier finding that this is the first Na+ dependent electron transfer step in the enzyme reaction. The results are discussed will be discussed in the context of our current model of Na+-NQR as a redox driven Na+ pump, that operates on the basis of kinetic rather than thermodynamic coupling.