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Probing the Active Site of an O2-Tolerant NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 by In Situ EPR and FTIR Spectroscopy

Authors

  • Marius Horch,

    1. Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin (Germany), Fax: (+49) 30-3142-1122
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    • These authors contributed equally to the work and are listed alphabetically.

  • Lars Lauterbach,

    1. Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin (Germany), Fax: (+49) 30-2093-8102
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    • These authors contributed equally to the work and are listed alphabetically.

  • Dr. Miguel Saggu,

    1. Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin (Germany), Fax: (+49) 30-3142-1122
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    • These authors contributed equally to the work and are listed alphabetically.

  • Prof. Peter Hildebrandt,

    1. Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin (Germany), Fax: (+49) 30-3142-1122
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  • Dr. Friedhelm Lendzian,

    1. Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin (Germany), Fax: (+49) 30-3142-1122
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  • Prof. Robert Bittl,

    1. Institut für Physik, Freie Universität Berlin, Arnimallee 14, 10115 Berlin (Germany)
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  • Dr. Oliver Lenz,

    1. Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin (Germany), Fax: (+49) 30-2093-8102
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  • Dr. Ingo Zebger

    1. Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin (Germany), Fax: (+49) 30-3142-1122
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  • The work was supported by the DFG (SFB498 and Cluster of Excellence “Unicat”). The authors are indebted to Bärbel Friedrich and Siem Albracht for critical comments and helpful discussions.

Abstract

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A clear picture: In situ EPR and FTIR spectroscopic studies on the soluble, NAD+-reducing [NiFe]-hydrogenase of Ralstonia eutropha reveal that the catalytic site resides predominantly in the intermediate Nia-C state within whole cells. This state, can either be reversibly oxidized to a “Nir-B”-like state or further reduced to various Nia-SR species. The data suggest that the iron center in the active site contains a standard set (one CO and two CN) of inorganic ligands.

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