Both Fdh-N and Fdh-O can catalyze

the formate-dependent reduction of either BV or DCPIP (2,6-dichlorophenolindophenol) [8, 9], whereby Fdh-N transfers electrons much more readily to DCPIP than to BV [8]. ICG-001 datasheet Analysis of fraction P1 from the gel filtration experiment revealed a formate: BV oxidoreductase activity of 67 mU mg protein-1 and a formate: DCPIP oxidoreductase activity of 0.64 U mg protein-1 (Table 1). In comparison, the H2: BV oxidoreductase activity of fraction P1 was 15 mU mg protein-1, while no enzyme activity could be detected when hydrogen gas was replaced with nitrogen gas. Table 1 Activity of enriched enzyme fraction with different electron donors Electron donor and acceptora Specific Activity (mU mg protein-1)b H2 and benzyl viologen 14.8 ± 2.3 Benzyl viologen without an electron donor < 0.20 Formate and benzyl viologen 1.24 ± 1.0 Formate and PMS/DCPIP 638.3 ± 69 a The buffer used was 50 mM sodium phosphate pH 7.2; BV was used at a final concentration of 4 mM; formate was added to a final concentration of 18 mM; and PMS/DCPIP were added at final concentrations of 20 μM and 78 μM, respectively. b The mean and standard click here deviation

(±) of at least three independent experiments are shown. All three Fdh enzymes in E. coli are selenocysteine-containing proteins [1, 2, 18]. Therefore, a mutant unable to incorporate selenocysteine co-translationally into the

polypeptides should lack this slow-migrating enzyme H2-oxidizing activity. Analysis of crude extracts derived from the selC mutant FM460, which is unable to synthesize the selenocysteine-inserting tRNASEC [19], lacked the hydrogenase-independent activity band observed in the wild-type (Figure 3), consistent with the activity being selenium-dependent. Notably Hyd-1 and Hyd-2 both retained activity in the selC mutant. Figure 3 A Chloroambucil selC mutant is devoid of the hydrogenase-independent H 2 : BV oxidoreductase activity. Extracts derived from MC4100 (lane 1) and the isogenic ΔselC mutant FM460 (lane 2) were separated by non-denaturing PAGE and subsequently stained for hydrogenase enzyme activity. Equivalent amounts of Triton X-100-treated crude extract (50 μg of protein) were applied to each lane. The activity bands corresponding to Hyd-1 and Hyd-2 are indicated, as is the activity band due to Fdh-N/Fdh-O (designated by an arrow). Fdh-N and Fdh-O can also transfer the electrons from hydrogen to other redox dyes The catalytic subunits of Fdh-N and Fdh-O are encoded by the fdnG and fdoG genes, respectively [5, 6]. To analyse the extent to which Fdh-N and Fdh-O contributed to hydrogen: BV oxidoreductase activity after fermentative growth the activity in mutants with a deletion mutation either in fdnG or in fdoG was analyzed.