Mechanism of Sulfide-Quinone Reductase Investigated Using Site-Directed Mutagenesis and Sulfur Analysis
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.
- Mechanism of Sulfide-Quinone Reductase Investigated Using Site-Directed Mutagenesis and Sulfur Analysis
- C. Griesbeck, M. Schutz, T. Schodl, S. Bathe, Prof. Dr. Lydia Nausch, N. Mederer, M. Vielreicher, G. Hauska
- Griesbeck, C.; Schutz, M.; Schodl, T.; Bathe, S.; Nausch, L.; Mederer, N.; Vielreicher, M.; Hauska, G. (2002): Mechanism of Sulfide-Quinone Reductase Investigated Using Site-Directed Mutagenesis and Sulfur Analysis. Biochemistry 41 (39), S. 11552-11565. DOI: 10.1021/bi026032b