Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12

Abstract

Hydrogenase isoenzyme 1 from the membrane fraction of anaerobically grown Escherichia coli has been purified to near homogeneity. The preparation involved dispersion of the membrane fraction with deoxycholate followed by ammonium sulphate precipitation, ion-exchange, hydroxyapatite and gel filtration chromatography steps. The enzyme was assayed by quantification of the H2 : benzyl viologen oxidoreductase activity immunoprecipitated by a non-inhibitory antiserum specific for the enzyme. The enzyme constituted about 8 % of the hydrogenase activity found in the detergent-dispersed membranes, the remainder being attributable to hydrogenase isoenzyme 2. Isoenzyme 1 was purified 130-fold and the specific activity of the final preparation was 10.6 pmol benzyl viologen reduced min-(mg protein)-' (H2 : benzyl viologen oxidoreductase). The final preparation contained polypeptides of apparent M , 64000, 31 000 and 29000. Antibodies were raised both to the final preparation and to immunoprecipitation arcs containing hydrogenase isoenzyme 1, excised from crossed immunoelectrophoresis plates. The former cross-reacted with all three polypeptides in the enzyme preparation but the latter recognised only the M,-64000 polypeptide. Immunological analysis revealed that the polypeptides of apparent M , 31000 and 29000 are fragments of a single polypeptide of M, 35000 which is present in the detergent-dispersed membranes. The fragmentation of the M,-35 000 polypeptide during the preparation correlates with a change in the electrophoretic mobility of the enzyme. A similar electrophoretic mobility change was observed, accompanied by cleavage of the Mr-35000 polypeptide to one of 32000 when the enzyme was analysed after exposure of detergent-dispersed membranes to trypsin. The enzyme in the detergent-dispersed membranes consists minimally of two subunits of M , 64000 and two subunits of M , 35000. It contained 12.2 mol Fe and 9.1 mol acid-labile S2-/200000 g enzyme. The enzyme, purified from bacteria grown in the presence of 63Ni, was found to contain 0.64 (+ 0.20) mol Ni/200 000 g enzyme. A constant ratio of 63Ni immunoprecipitated to hydrogenase isoenzyme 1 activity immunoprecipitated by antiserum specific for the enzyme was observed during the preparation, consistent with Ni being part of the enzyme. The enzyme has a low K , for H2 (2.0 pM) in the H2 : benzyl viologen oxidoreductase assay. It catalyses H2 evolution employing reduced methyl viologen as electron donor. It is inhibited reversibly by CO and irreversibly by N-bromosuccinimide.Hydrogenase (EC 1.12. -. -) in Escherichia coli is involved in two distinct modes of H2 metabolism [l]. The formate hydrogenlyase system catalyses the oxidation of endogenously produced formate to C 0 2 and H2 during anaerobic growth on glucose in the absence of a terminal respiratory electron acceptor. An hydrogenase forms part of the formate hydrogenlyase system [2]. E. coli is also able to derive energy to support anaerobic growth from the oxidation of H2 coupled to the reduction of fumarate, a non...

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

“…Since an internal redox state characterized by a low NADH/NAD þ ratio could promote the activity of these pathways, we hypothesize that such a redox state is created in DfdhF by the lack of hydrogen generation and recycling. The metabolic recycling of the hydrogen produced by FHL is known to increase the NADH/NAD þ ratio, thus promoting the synthesis of reduced products (Clark, 1989;Sawers and Boxer, 1986;Sawers et al, 1985Murarka et al, 2008).…”

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

“…Aliquots of protein (25 mg) from crude extracts were separated by SDS-PAGE in 10 % (w/v) gels (Laemmli, 1970) and transferred to nitrocellulose membranes as described by Towbin et al (1979). Antibodies raised against Hyd-1 (1 : 10 000; Sawers & Boxer, 1986), Hyd-2 (1 : 20 000), HycE (1 : 3000; Sauter et al, 1992) and PflB (1 : 3000; Sawers et al, 1998) were used. Secondary antibody conjugated to horseradish peroxidase was obtained from Bio-Rad.…”

The CO and CN− ligands to the active site Fe in [NiFe]‐hydrogenase of Escherichia coli have different metabolic origins

“…Hydrogenase 1 (Hyd-1) and Hyd-2 function in anaerobic hydrogen oxidation, while Hyd-3 forms part of the multi-subunit formate hydrogenlyase complex, which catalyzes H 2 evolution during fermentation. Only Hyd-1 and Hyd-2 have been characterised biochemically [3,4].…”