A similar route has been demonstrated for the selenate-respiring bacterium T. selenatis (Lowe et al., 2010). In T. selenatis, electrons are mediated between membrane-bound quinol:cytochrome c oxidoreductase (bc1-complex) and periplasmic selenate reductase (Ser) by the 24-kDa di-heme cytochrome cytc-Ts4. In the photosynthetic bacterium R. sulfidophilum, electrons are transferred in the opposite direction on the oxidation of dimethyl sulfide to DMSO. Electrons

are shuttled from the periplasmic DMS dehydrogenase to the membrane-bound photosynthetic reaction center, mediated by the soluble cytochrome c2 (Creevey et al., 2008). In the present paper, we describe the purification and characterization of the cytochrome c discussed above. MS gives a molecular weight of about 9 kDa rather than selleck compound the 6 kDa found by SDS-PAGE. We denote this protein cytochrome c-Id1. Electron transfer to chlorate is thermodynamically feasible from its EX 527 concentration estimated redox potential, and we demonstrate its ability to serve as electron donor for purified chlorate reductase. Ideonella dechloratans was obtained from the Culture Collection of Göteborg University, Göteborg, Sweden. Cells were cultured anaerobically (8 L) and harvested by centrifugation at 8000 g for 15 min. Wet

cells (20 g) were resuspended in 90 mL of 0.3 M Tris-HCl, pH 8.1, containing 20% (w/v) sucrose and 1 mM EDTA. The suspension was placed at room temperature for 10 min, followed by centrifugation at 8000 g for 10 min. The pellet was resuspended in 90 mL ice-cold 0.5 mM MgCl, and was kept on ice for 10 min.

Soluble proteins were extracted by from centrifugation at 8000 g for 20 min and ammonium sulfate was added to the protein extract to 40% saturation. The solution was stirred on ice for 30 min, followed by centrifugation at 18 000 g for 10 min. Ammonium sulfate was added to the supernatant to 85% saturation and the solution was stirred on ice for 30 min. Precipitated proteins were pelleted by centrifugation at 18 000 g for 10 min, and resuspended in 10 mL sodium phosphate (50 mM, pH 7.0) containing ammonium sulfate (0.92 M). The solution was applied on to a Phenyl Sepharose 6 Fast Flow (low sub) column, 20 × 2.6 cm (GE Healthcare, Uppsala, Sweden) at a flow rate of 1 mL min−1. The column was washed with 60 mL sodium phosphate/ammonium sulfate (50 mM/0.92 M, pH 7.0) and was eluted using a gradient of 500 mL (0.92–0 M ammonium sulfate) at a flow rate of 2 mL min−1. The cytochrome c was eluted at approximately 0.37 M ammonium sulfate. Appropriate fractions were pooled and concentrated using an Amicon stirred cell 8050 with Ultrafiltration Membrane, MWCO 1000 Da (Millipore, Solna, Sweden). The concentrated sample was applied on to a Sephacryl S-200 Hiprep™ 16/60 column (GE Healthcare) and eluted using 0.1 M sodium phosphate, pH 7.0, with the flow rate of 0.1 mL min−1.