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 oxidoreduct ase (Na+-NQR) from Vibrio alginolyticus was investigated by ultraviole t-visible and fluorescence spectroscopy. The reduction of Na+-NQR by e xcess 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, respectivel y, but the fully reduced (Fl(red)H(2)) or oxidized flavin (Fl(ox)) pre vailed. During reoxidation of Fl(red)H(2) with Q-1, the intermediate f ormation of an absorbance band around 800 nm was observed, which was t entatively assigned as the Fl(red)H(-)-NAD(+) charge-transfer complex. Complete reoxidation of Fl(red)H(2) in Na+-NQR was achieved by a five fold excess of Q-1 over NADH. These results indicated that only a smal l fraction of FAD was in the flavosemiquinone redox state during turno ver 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 s pectra of FAD (Fl(ox)). The addition of Ag+ resulted in a marked incre ase 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 wit h the loss of enzyme activity. Gel filtration of the Ag+-treated Na+-N QR confirmed that FAD had been displaced from the hole-enzyme.