title: O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase.
abstract: Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ∼50% decrease of the number of ∼2.7-Å Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.
authors.affiliation: Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
authors.forename: Camilla
authors.initials: C
authors.lastname: Lambertz
authors.forename: Nils
authors.initials: N
authors.lastname: Leidel
authors.forename: Kajsa G V
authors.initials: KG
authors.lastname: Havelius
authors.forename: Jens
authors.initials: J
authors.lastname: Noth
authors.forename: Petko
authors.initials: P
authors.lastname: Chernev
authors.forename: Martin
authors.initials: M
authors.lastname: Winkler
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology*; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy
chemical_list: D007506:Iron-Sulfur Proteins; D017382:Reactive Oxygen Species; D007501:Iron; C118830:iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen