The Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus - Redox states of the FAD prosthetic group and mechanism of Ag+ inhibition

The FAD prosthetic group of the Na+-motive NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio alginolyticus was investigated by ultraviolet-visible and fluorescence spectroscopy. The reduction of Na+-NQR by excess NADH in the presence of 6-13 mu M O-2 resulted in the formation of the blue flavosemiquinone radical. If the concentration of dioxygen was further reduced to 0.1 mu M O-2, neither the reduction of Na+-NQR by NADH nor its reoxidation with ubiquinone-1 (Q-1) yielded a stable flavosemiquinone in equilibrium with reductant or oxidant, respectively, but the fully reduced (Fl(red)H(2)) or oxidized flavin (Fl(ox)) prevailed. During reoxidation of Fl(red)H(2) with Q-1, the intermediate formation of an absorbance band around 800 nm was observed, which was tentatively assigned as the Fl(red)H(-)-NAD(+) charge-transfer complex. Complete reoxidation of Fl(red)H(2) in Na+-NQR was achieved by a fivefold excess of Q-1 over NADH. These results indicated that only a small fraction of FAD was in the flavosemiquinone redox state during turnover to mediate the electron transfer between the hydride donor, NADH, and the one-electron acceptor [2Fe-2S]. The titration of Na+-NQR with Ag+, a specific inhibitor, was followed by the fluorescence emission spectra of FAD (Fl(ox)). The addition of Ag+ resulted in a marked increase of the flavin fluorescence (16% at 200 nM Ag+), with half-maximal saturation at approximately 50 nM Ag+, indicating dissociation of FAD from the enzyme. The increase in fluorescence intensity correlated with the loss of enzyme activity. Gel filtration of the Ag+-treated Na+-NQR confirmed that FAD had been displaced from the hole-enzyme.