Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae.
The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen Vibrio cholerae is a respiratory flavo-FeS complex composed of the six subunits NqrA-F. The Na(+)-NQR was produced as His(6)-tagged protein by homologous expression in V. cholerae. The isolated complex contained near-stoichiometric amounts of non-covalently bound FAD (0.78 mol/mol Na(+)-NQR) and riboflavin (0.70 mol/mol Na(+)-NQR), catalyzed NADH-driven Na(+) transport (40 nmol Na(+)min(-1) mg(-1)), and was inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide. EPR spectroscopy showed that Na(+)-NQR as isolated contained very low amounts of a neutral flavosemiquinone (10(-3) mol/mol Na(+)-NQR). Reduction with NADH resulted in the formation of an anionic flavosemiquinone (0.10 mol/mol Na(+)-NQR). Subsequent oxidation of the Na(+)-NQR with ubiquinone-1 or O(2) led to the formation of a neutral flavosemiquinone (0.24 mol/mol Na(+)-NQR). We propose that the Na(+)-NQR is fully oxidized in its resting state, and discuss putative schemes of NADH-triggered redox transitions.
Tao
Minli
M
Biochemisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.
Casutt
Marco S
MS
Fritz
Günter
G
Steuber
Julia
J
D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011808:Quinone Reductases / Q000235:genetics / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013053:Spectrophotometry; D014734:Vibrio cholerae / Q000201:enzymology*
D001426:Bacterial Proteins; D017931:DNA Primers; D005415:Flavins; D016877:Oxidants; D011809:Quinones; D011994:Recombinant Proteins; C001636:flavosemiquinone; D005182:Flavin-Adenine Dinucleotide; D012964:Sodium; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D012256:Riboflavin