The Conformational Changes Induced by Ubiquinone Binding in the Na+-pumping NADH:Ubiquinone Oxidoreductase (Na+-NQR) Are Kinetically Controlled by Conserved Glycines 140 and 141 of the NqrB Subunit

Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

“…9A) to reflect a perturbed but still functioning electron transfer step between the two sites (10). It is worth noting that the same FTIR spectroelectrochemical study also found evidence for structural differences in the environment surrounding a bound inhibitor (HQNO) between the wild-type enzyme and the mutants (NqrB-G140A and -G141V) (10). Thus, it appears likely that in the mutants the structural rearrangement of NqrB, due to the reduction of cofactors, may be able to take place even in the presence of bound inhibitor.…”

“…In subsequent studies (8,10), working with the V. cholerae Na ϩ -NQR, Barquera and co-workers demonstrated important functional roles for NqrB-Gly-140 and -Gly-141 in the electron transfer to ubiquinone. However, the effects of mutations at these positions on the inhibition of Na ϩ - We have now examined changes in the inhibitor sensitivity of five mutant enzymes, three with substitutions in helix II of NqrB, near the spontaneous mutation identified by Hayashi et al (15) (G140A, G141V, and E144C in the NqrB subunit), and two at the rear wall of the putative ubiquinone-binding cavity (Y36A and G38V in the NqrA subunit), as described above, using korormicin, aurachin D-42, PAD-1, PAD-2, and HQNO ( Table 1).…”

“…Na ϩ -NQR that has been purified using the non-ionic detergent dodecyl maltoside (DDM) is reported to contain tightly bound ubiquinone-8 (Q 8 ) in substoichiometric quantities that can be removed by washing the enzyme preparation with a buffer containing the zwitterionic detergent lauryldimethylamine N-oxide (LDAO) (7,10). We used LDAO-washed Na ϩ -NQR throughout this study.…”

“…With the two strong inhibitors in hand, we have used a photoaffinity labeling technique to identify the binding sites for ubiquinone and aurachintype inhibitors in isolated V. cholerae Na ϩ -NQR. We also investigated the mechanism of inhibition of aurachin derivatives using the mutated enzymes G140A, G141V, and E144C in the NqrB subunit, in which the effects of these inhibitors are significantly attenuated (8,10,15). Some of the current results were initially difficult to reconcile.…”

“…Because the work by Tuz et al (14) was largely based on steadystate kinetic analysis of mutants, it is difficult to determine whether the critical residues that they identified (NqrB-Phe-211 and -Phe-213) are directly involved in forming the binding pocket for the ubiquinone ring or whether they affect the reaction of ubiquinone indirectly though some long-range conformational change. In addition, to determine the number of binding sites and/or the dissociation constant for short-chain ubiquinone and the inhibitor HQNO, previous studies employed the equilibrium dialysis method with isolated wild-type and mutated Na ϩ -NQR (10,14). However, interpretation of data obtained through this method can be problematic because it may be impossible to distinguish between specific and non-specific binding of the hydrophobic ligands to the enzyme, particularly in the case of an integral membrane protein.…”