title: Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis.
abstract: Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.
authors.affiliation: Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Griesbeck
authors.forename: Michael
authors.initials: M
authors.lastname: Schütz
authors.forename: Thomas
authors.initials: T
authors.lastname: Schödl
authors.forename: Stephan
authors.initials: S
authors.lastname: Bathe
authors.forename: Lydia
authors.initials: L
authors.lastname: Nausch
authors.forename: Nicola
authors.initials: N
authors.lastname: Mederer
authors.forename: Martin
authors.initials: M
authors.lastname: Vielreicher
authors.forename: Günter
authors.initials: G
authors.lastname: Hauska
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016227:Benzoquinones / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004579:Electron Transport / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D013431:Sulfates / Q000032:analysis; D013440:Sulfides / Q000032:analysis; D013455:Sulfur / Q000032:analysis*; D014633:Valine / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D016227:Benzoquinones; D011993:Recombinant Fusion Proteins; D013431:Sulfates; D013440:Sulfides; C012036:sodium sulfate; D001224:Aspartic Acid; C004532:quinone; D006639:Histidine; D013455:Sulfur; C032915:polysulfide; D011808:Quinone Reductases; C073412:sulfide quinone reductase; D014633:Valine; D000409:Alanine