Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16.
This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.
Saggu
Miguel
M
From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin.
Zebger
Ingo
I
From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin. Electronic address: ingo.zebger@tu-berlin.de.
Ludwig
Marcus
M
Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
Lenz
Oliver
O
Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
Friedrich
Bärbel
B
Institute of Biology, Department of Microbiology, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
Hildebrandt
Peter
P
From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin.
Lendzian
Friedhelm
F
From the Institut für Chemie, Technische Universität Berlin, PC14, Strasse des 17. Juni 135, D-10623 Berlin. Electronic address: f.lendzian@tu-berlin.de.
D002384:Catalysis; D002392:Catechin / Q000378:metabolism; D020118:Cupriavidus necator / Q000201:enzymology*; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010957:Plasmids; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared
D008565:Membrane Proteins; D009532:Nickel; D002392:Catechin; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen