title: Resonance Raman Spectroscopic Analysis of the [NiFe] Active Site and the Proximal [4Fe-3S] Cluster of an O2-Tolerant Membrane-Bound Hydrogenase in the Crystalline State.
abstract: We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Nir-B [Ni(III)] and Nir-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Nia-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Nia-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H2-reduced active site states.
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Siebert
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Rippers
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Frielingsdorf
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Fritsch
authors.affiliation: Charité - Universitätsmedizin Berlin , Institut für Medizinische Physik und Biophysik (CC2), Group Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Charité - Universitätsmedizin Berlin , Institut für Medizinische Physik und Biophysik (CC2), Group Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Kalms
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Sagie
authors.initials: S
authors.lastname: Katz
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.affiliation: Charité - Universitätsmedizin Berlin , Institut für Medizinische Physik und Biophysik (CC2), Group Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Scheerer
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Paasche
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. C7, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Pelmenschikov
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Kuhlmann
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Maria Andrea
authors.initials: MA
authors.lastname: Mroginski
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Zebger
authors.affiliation: Technische Universität Berlin, Institut für Chemie , Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
mesh_terms: D020134:Catalytic Domain; D003460:Crystallization; D020118:Cupriavidus necator / Q000201:enzymology; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011789:Quantum Theory; D013059:Spectrum Analysis, Raman
chemical_list: D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen