title: The Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus--redox states of the FAD prosthetic group and mechanism of Ag+ inhibition.
abstract: 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 microM O2 resulted in the formation of the blue flavosemiquinone radical. If the concentration of dioxygen was further reduced to 0.1 microM O2, 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)H2) or oxidized flavin (Fl(ox)) prevailed. During reoxidation of Fl(red)H2 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)H2 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 holo-enzyme.
authors.affiliation: ETH-Zentrum, Zürich, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Steuber
authors.forename: W
authors.initials: W
authors.lastname: Krebs
authors.forename: P
authors.initials: P
authors.lastname: Dimroth
mesh_terms: D001426:Bacterial Proteins; D001692:Biological Transport; D002850:Chromatography, Gel; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism; D012834:Silver / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014451:Ubiquinone / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011809:Quinones; C001636:flavosemiquinone; D009243:NAD; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; D012834:Silver; D012964:Sodium; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases; D009245:NADH Dehydrogenase; D010100:Oxygen