title: Hydride binding to the active site of [FeFe]-hydrogenase.
abstract: [FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases.
authors.affiliation: Institute for Experimental Physics, Free University Berlin , 14195 Berlin, Germany.
authors.forename: Petko
authors.initials: P
authors.lastname: Chernev
authors.forename: Camilla
authors.initials: C
authors.lastname: Lambertz
authors.forename: Annika
authors.initials: A
authors.lastname: Brünje
authors.forename: Nils
authors.initials: N
authors.lastname: Leidel
authors.forename: Kajsa G V
authors.initials: KG
authors.lastname: Sigfridsson
authors.forename: Ramona
authors.initials: R
authors.lastname: Kositzki
authors.forename: Chung-Hung
authors.initials: CH
authors.lastname: Hsieh
authors.forename: Shenglai
authors.initials: S
authors.lastname: Yao
authors.forename: Rafael
authors.initials: R
authors.lastname: Schiwon
authors.forename: Matthias
authors.initials: M
authors.lastname: Driess
authors.forename: Christian
authors.initials: C
authors.lastname: Limberg
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D020134:Catalytic Domain; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D003486:Cyanides / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D056928:X-Ray Absorption Spectroscopy
chemical_list: D003486:Cyanides; D005296:Ferrous Compounds; D007506:Iron-Sulfur Proteins; D002248:Carbon Monoxide; D006859:Hydrogen; C118830:iron hydrogenase; D006864:Hydrogenase