title: CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase.
abstract: Hydrogenases are nature's key catalysts involved in both microbial consumption and production of molecular hydrogen. H2exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H2, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry-that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H2oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O2, employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N10-formyl-tetrahydrofolate (N10-CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N10-CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates.
authors.affiliation: Department of Biology, Microbiology, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.|Department of Chemistry, Biophysical Chemistry, Technische Universität Berlin, 10623 Berlin, Germany.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Bürstel
authors.affiliation: Department of Chemistry, Biophysical Chemistry, Technische Universität Berlin, 10623 Berlin, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Siebert
authors.affiliation: Department of Biology, Microbiology, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.|Department of Chemistry, Biophysical Chemistry, Technische Universität Berlin, 10623 Berlin, Germany.
authors.forename: Stefan
authors.identifier: 0000-0002-4141-7836
authors.initials: S
authors.lastname: Frielingsdorf
authors.affiliation: Department of Chemistry, Biophysical Chemistry, Technische Universität Berlin, 10623 Berlin, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.affiliation: Department of Biology, Microbiology, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.affiliation: Department of Biology, Microbiology, Humboldt-Universität zu Berlin, 10115 Berlin, Germany; oliver.lenz@tu-berlin.de.|Department of Chemistry, Biophysical Chemistry, Technische Universität Berlin, 10623 Berlin, Germany.
authors.forename: Oliver
authors.identifier: 0000-0003-4550-5128
authors.initials: O
authors.lastname: Lenz
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D002244:Carbon / Q000737:chemistry* / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D020118:Cupriavidus necator; D017931:DNA Primers; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013997:Time Factors
chemical_list: D017931:DNA Primers; D008024:Ligands; D000244:Adenosine Diphosphate; D002244:Carbon; D002248:Carbon Monoxide; D006859:Hydrogen; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D005998:Glycine
keywords: carbonyl ligand; formyl-THF; hydrogenase; metalloenzyme; one-carbon metabolism