title: Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics.
abstract: The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN(-)) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (13)CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN(-) at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (13)CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.
authors.affiliation: Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany;
authors.forename: Moritz
authors.initials: M
authors.lastname: Senger
authors.affiliation: Department of Physics, Biophysics of Metalloenzymes, Freie Universität Berlin, 14195 Berlin, Germany;
authors.forename: Stefan
authors.initials: S
authors.lastname: Mebs
authors.affiliation: Department of Biochemistry of Plants, Photobiotechnology, Ruhr-Universität Bochum, 44801 Bochum, Germany;
authors.forename: Jifu
authors.initials: J
authors.lastname: Duan
authors.affiliation: Department of Chemistry and Biochemistry, Inorganic Chemistry I, Ruhr-Universität Bochum, 44801 Bochum, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Wittkamp
authors.affiliation: Department of Chemistry and Biochemistry, Inorganic Chemistry I, Ruhr-Universität Bochum, 44801 Bochum, Germany.
authors.forename: Ulf-Peter
authors.initials: UP
authors.lastname: Apfel
authors.affiliation: Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany;
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
authors.affiliation: Department of Physics, Biophysics of Metalloenzymes, Freie Universität Berlin, 14195 Berlin, Germany;
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
authors.affiliation: Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany; sven.stripp@fu-berlin.de.
authors.forename: Sven Timo
authors.initials: ST
authors.lastname: Stripp
mesh_terms: D020418:Algal Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D002247:Carbon Isotopes / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D003013:Clostridium / Q000201:enzymology; D003486:Cyanides / Q000378:metabolism; D045285:Desulfovibrio desulfuricans / Q000201:enzymology; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D020418:Algal Proteins; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D003486:Cyanides; D008024:Ligands; D002248:Carbon Monoxide; D006859:Hydrogen; D007501:Iron; D006864:Hydrogenase
keywords: [FeFe]-hydrogenase; cofactor dynamics; density functional theory; infrared spectroscopy; isotope editing