caa a22 4500 605506973 CHVBK 20210128100629.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00775-014-1203-9 doi (NATIONALLICENCE)springer-10.1007/s00775-014-1203-9 Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states [Elektronische Daten] [Anne Volbeda, Lydie Martin, Elodie Barbier, Oscar Gutiérrez-Sanz, Antonio De Lacey, Pierre-Pol Liebgott, Sébastien Dementin, Marc Rousset, Juan Fontecilla-Camps] Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-‘Sox' species. SBIC, 2014 Ni-Fe active site nationallicence Oxygen-sensitivity nationallicence Ni-A state nationallicence Ni-SU state nationallicence Ni-‘Sox' state nationallicence Av : Allochromatium vinosum nationallicence Df : Desulfovibrio fructosovorans nationallicence Dv H : Desulfovibrio vulgaris Hildenborough nationallicence Dv MF : Desulfovibrio vulgaris Miyazaki F nationallicence MSR : Methionine sulfoxide reductase nationallicence WT : Wild type nationallicence Volbeda Anne University Grenoble Alpes, IBS, 38044, Grenoble, France aut Martin Lydie University Grenoble Alpes, IBS, 38044, Grenoble, France aut Barbier Elodie University Grenoble Alpes, IBS, 38044, Grenoble, France aut Gutiérrez-Sanz Oscar Instituto de Catálisis y Petroleoquímica, CSIC, 28049, Madrid, Spain aut De Lacey Antonio Instituto de Catálisis y Petroleoquímica, CSIC, 28049, Madrid, Spain aut Liebgott Pierre-Pol Aix-Marseille Université, CNRS, IMM, 13402, Marseille, France aut Dementin Sébastien Aix-Marseille Université, CNRS, IMM, 13402, Marseille, France aut Rousset Marc Aix-Marseille Université, CNRS, IMM, 13402, Marseille, France aut Fontecilla-Camps Juan University Grenoble Alpes, IBS, 38044, Grenoble, France aut JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/1(2015-01-01), 11-22 0949-8257 20:1<11 2015 20 775 https://doi.org/10.1007/s00775-014-1203-9 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00775-014-1203-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Volbeda Anne University Grenoble Alpes, IBS, 38044, Grenoble, France aut NATIONALLICENCE 700 1- Martin Lydie University Grenoble Alpes, IBS, 38044, Grenoble, France aut NATIONALLICENCE 700 1- Barbier Elodie University Grenoble Alpes, IBS, 38044, Grenoble, France aut NATIONALLICENCE 700 1- Gutiérrez-Sanz Oscar Instituto de Catálisis y Petroleoquímica, CSIC, 28049, Madrid, Spain aut NATIONALLICENCE 700 1- De Lacey Antonio Instituto de Catálisis y Petroleoquímica, CSIC, 28049, Madrid, Spain aut NATIONALLICENCE 700 1- Liebgott Pierre-Pol Aix-Marseille Université, CNRS, IMM, 13402, Marseille, France aut NATIONALLICENCE 700 1- Dementin Sébastien Aix-Marseille Université, CNRS, IMM, 13402, Marseille, France aut NATIONALLICENCE 700 1- Rousset Marc Aix-Marseille Université, CNRS, IMM, 13402, Marseille, France aut NATIONALLICENCE 700 1- Fontecilla-Camps Juan University Grenoble Alpes, IBS, 38044, Grenoble, France aut NATIONALLICENCE 773 0- JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/1(2015-01-01), 11-22 0949-8257 20:1<11 2015 20 775