Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron–sulfur cluster-containing small subunit

Abstract

Escherichia coli can both oxidize hydrogen and reduce protons. These activities involve three distinct [NiFe]-hydrogenases, termed Hyd-1, Hyd-2, and Hyd-3, each minimally comprising heterodimers of a large subunit, containing the [NiFe] active site, and a small subunit, bearing iron-sulfur clusters. Dihydrogen-oxidizing activity can be determined using redox dyes like benzyl viologen (BV); however, it is unclear whether electron transfer to BV occurs directly at the active site, or via an iron-sulfur center in the small subunit. Plasmids encoding Strep-tagged derivatives of the large subunits of the three E. coli [NiFe]-hydrogenases restored activity of the respective hydrogenase to strain FTD147, which carries in-frame deletions in the hyaB, hybC, and hycE genes encoding the large subunits of Hyd-1, Hyd-2, and Hyd-3, respectively. Purified Strep-HyaB was associated with the Hyd-1 small subunit (HyaA), and purified Strep-HybC was associated with the Hyd-2 small subunit (HybO), and a second iron-sulfur protein, HybA. However, Strep-HybC isolated from a hybO mutant had no other associated subunits and lacked BV-dependent hydrogenase activity. Mutants deleted separately for hyaA, hybO, or hycG (Hyd-3 small subunit) lacked BV-linked hydrogenase activity, despite the Hyd-1 and Hyd-2 large subunits being processed. These findings demonstrate that hydrogenase-dependent reduction of BV requires the small subunit.

Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12

“…Under those conditions, about 90 % of the activity is due to Hyd-3 and only 1 % is due to Hyd-1 (Pinske et al, 2011). This correlated with the appearance of hydrogen gas measured qualitatively, due to the activity of the hydrogenevolving FHL complex (data not shown).…”

“…Recombinant DNA work was carried out according to published methods (Sambrook & Russell, 2001). Cloning of the hyaA upstream regulatory region for W(hyaA9-9lacZ) translational fusion analysis has been described (Pinske & Sawers, 2011). For the W(hycA-lacZ) transcriptional fusion construction plasmid pTS101 (Schlensog et al, 1994) was used, and the fusion was transferred to lRS45 (Simons et al, 1987) and introduced in single copy into the l attachment site of MC4100 resulting in strain CP782.…”

[NiFe]‐hydrogenase maturation: Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

“…Non-denaturing polyacrylamide gel electrophoresis (PAGE) using 7.5% (w/v) polyacrylamide with subsequent staining for hydrogenase enzyme activity was performed exactly as described [25]. SDS-PAGE was performed using 15% (w/v) polyacrylamide as described [26].…”

“…To demonstrate that the mutation caused inactivation of HypD, the resulting plasmid, pT-hypDEFCStrep[C41A] was transformed into the hypD mutant DHP-D [9] and the ability of HypD C41A to restore Hyd-1 and Hyd-2 enzyme activity to the mutant was monitored by enzyme-specific activity staining (Fig. 3) [25]. It should be noted that in MC4100 the hyp genes on pT-hypDEFCStrep are expressed due to inherent promoter activity on the plasmid backbone.…”

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

“…Cells were harvested, washed and 100 mg of cells re-suspended in 200 μL of 50 mM Tris.HCl. Hydrogen oxidising activity was measured in cuvettes containing H2-saturated buffer and the artificial electron acceptor benzyl viologen [36]. The reaction was started by the addition of intact cells and H 2 dependent benzyl viologen reduction was measured at an absorbance of 600 nm.…”