title: Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.
abstract: During mixed-acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO2and H2. Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO2to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function.
authors.affiliation: Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, Scotland, DD1 5EH, United Kingdom.
authors.forename: Constanze
authors.initials: C
authors.lastname: Pinske
authors.affiliation: Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, Scotland, DD1 5EH, United Kingdom. f.sargent@dundee.ac.uk.
authors.forename: Frank
authors.identifier: http://orcid.org/0000-0002-6841-3624
authors.initials: F
authors.lastname: Sargent
mesh_terms: D000595:Amino Acid Sequence; D002245:Carbon Dioxide / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology; D005560:Formate Dehydrogenases / Q000378:metabolism*; D005561:Formates / Q000737:chemistry*; D006864:Hydrogenase / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D005561:Formates; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C030544:formic acid; D002245:Carbon Dioxide; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase
keywords: [NiFe] hydrogenase; bacterial hydrogen metabolism; formate chemosynthesis; formate dehydrogenase; formate hydrogenlyase; site-directed mutagenesis