Bioenergetics of the staphylococcal multidrug export protein QacA. Identification of distinct binding sites for monovalent and divalent cations.

The multidrug efflux pump QacA from Staphylococcus aureus confers resistance to an extensive range of structurally dissimilar compounds. Fluorimetric analyses demonstrated that QacA confers resistance to the divalent cation 4',6-diamidino-2-phenylindole, utilizing a proton motive force-dependent efflux mechanism previously demonstrated for QacA-mediated resistance to the monovalent cation ethidium. Both the ionophores nigericin and valinomycin inhibited QacA-mediated export of ethidium, indicating an electrogenic drug/nH+ (n >/= 2) antiport mechanism. The kinetic parameters, Km and Vmax, were determined for QacA-mediated export of four fluorescent substrates, 4',6-diamidino-2-phenylindole, 3', 3'-dipropyloxacarbocyanine, ethidium, and pyronin Y. Competition studies showed that QacA-mediated ethidium export is competitively inhibited by monovalent cations, e.g. benzalkonium, and non-competitively inhibited by divalent cations, e.g. propamidine, which suggests that monovalent and divalent cations bind at distinct sites on the QacA protein. The quaternary ammonium salt, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, was used as a membrane-specific fluorescence probe and demonstrated that the amount of substrate entering the inner leaflet was significantly reduced in QacA-containing strains, supporting the notion that the substrate is extruded directly from the membrane.