title: Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stable-isotope labeling.
abstract: [Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized.
authors.affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse 10, 35043 Marburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schick
authors.forename: Xiulan
authors.initials: X
authors.lastname: Xie
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ataka
authors.forename: Jörg
authors.initials: J
authors.lastname: Kahnt
authors.forename: Uwe
authors.initials: U
authors.lastname: Linne
authors.forename: Seigo
authors.initials: S
authors.lastname: Shima
mesh_terms: D000085:Acetates / Q000737:chemistry; D001105:Archaea / Q000378:metabolism*; D002244:Carbon / Q000737:chemistry; D002245:Carbon Dioxide / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008697:Methane / Q000737:chemistry*; D008956:Models, Chemical; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013055:Spectrophotometry, Infrared / Q000379:methods
chemical_list: D000085:Acetates; D002247:Carbon Isotopes; D007506:Iron-Sulfur Proteins; D008024:Ligands; D011522:Protons; D002245:Carbon Dioxide; D002244:Carbon; D007501:Iron; C118830:iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine; D008697:Methane