title: Characterization of the kinetics and electron paramagnetic resonance spectroscopic properties of Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR).
abstract: Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR) catalyzes the oxidation of sulfide to polysulfide chains or elemental sulfur coupled to quinone reduction via a non-covalent FAD cofactor. We investigated the role of the FAD using kinetics and EPR spectroscopy. The properties of the enzyme were compared with alanine and/or serine variants of conserved cysteine residues (Cys128, Cys160, Cys356) structurally close to the FAD cofactor and histidine residues (His132, His198) implicated in function. When the pre-steady state reduction of FAD was monitored, variants of Cys128 and His132 had similar rates to wild-type enzyme confirming they do not participate in the reductive half reaction whereas variants of Cys160, Cys356 and His198 had greatly reduced activity. Using steady state kinetics of Na2S-dependent decylubiquinone (DUQ) reduction we measured a kcat of 6.5s(-1) and a Km (Na2S) of 3.0μM and a Km (DUQ) of 3.4μM. Variants of Cys160, Cys356 and His198 had greatly diminished DUQ reduction activity whereas variants of Cys128 and His132 were less affected. A neutral flavin semiquinone was observed in the EPR spectrum of SQR reduced with Na2S which was enhanced in the Cys160Ala variant suggesting the presence of a Cys356-S(γ)-S-C(4A)-FAD adduct. Potentiometric titrations of the FAD semiquinone revealed an Em of -139±4mV at pH 7.0.
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Electronic address: joel.weiner@ualberta.ca.
authors.forename: Joel H
authors.initials: JH
authors.lastname: Weiner
mesh_terms: D042763:Acidithiobacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; D011808:Quinone Reductases; C073412:sulfide quinone reductase
keywords: Flavin radical; Flavoprotein; Oxidative respiration; Ubiquinone