Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis
| Autor: | Lydia Nausch, Günter Hauska, Martin Vielreicher, Thomas Schödl, Stephan Bathe, Christoph Griesbeck, Nicola Mederer, Michael Schütz |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Sulfide
Recombinant Fusion Proteins Molecular Sequence Data Flavoprotein chemistry.chemical_element Sulfides Quinone oxidoreductase Biochemistry Substrate Specificity Serine Electron Transport Bacterial Proteins Benzoquinones Histidine Amino Acid Sequence Quinone Reductases Site-directed mutagenesis Chromatography High Pressure Liquid chemistry.chemical_classification Aspartic Acid Rhodobacter Alanine biology Sulfates Valine Gene Expression Regulation Bacterial biology.organism_classification Sulfur Enzyme Activation Spectrometry Fluorescence chemistry Amino Acid Substitution biology.protein Mutagenesis Site-Directed Oxidation-Reduction Cysteine |
| Zdroj: | Biochemistry. 41(39) |
| ISSN: | 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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