title: EPR and FTIR analysis of the mechanism of H2 activation by [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii.
abstract: While a general model of H2 activation has been proposed for [FeFe]-hydrogenases, the structural and biophysical properties of the intermediates of the H-cluster catalytic site have not yet been discretely defined. Electron paramagnetic resonance (EPR) spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the H-cluster catalytic site, a [4Fe-4S]H subcluster linked by a cysteine thiolate to an organometallic diiron subsite with CO, CN, and dithiolate ligands, in [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii (CrHydA1). Oxidized CrHydA1 displayed a rhombic 2.1 EPR signal (g = 2.100, 2.039, 1.997) and an FTIR spectrum previously assigned to the oxidized H-cluster (Hox). Reduction of the Hox sample with 100% H2 or sodium dithionite (NaDT) nearly eliminated the 2.1 signal, which coincided with appearance of a broad 2.3-2.07 signal (g = 2.3-2.07, 1.863) and/or a rhombic 2.08 signal (g = 2.077, 1.935, 1.880). Both signals displayed relaxation properties similar to those of [4Fe-4S] clusters and are consistent with an S = 1/2 H-cluster containing a [4Fe-4S]H(+) subcluster. These EPR signals were correlated with differences in the CO and CN ligand modes in the FTIR spectra of H2- and NaDT-reduced samples compared with Hox. The results indicate that reduction of [4Fe-4S]H from the 2+ state to the 1+ state occurs during both catalytic H2 activation and proton reduction and is accompanied by structural rearrangements of the diiron subsite CO/CN ligand field. Changes in the [4Fe-4S]H oxidation state occur in electron exchange with the diiron subsite during catalysis and mediate electron transfer with either external carriers or accessory FeS clusters.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United States.
authors.forename: David W
authors.initials: DW
authors.lastname: Mulder
authors.forename: Michael W
authors.initials: MW
authors.lastname: Ratzloff
authors.forename: Eric M
authors.initials: EM
authors.lastname: Shepard
authors.forename: Amanda S
authors.initials: AS
authors.lastname: Byer
authors.forename: Seth M
authors.initials: SM
authors.lastname: Noone
authors.forename: John W
authors.initials: JW
authors.lastname: Peters
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
authors.forename: Paul W
authors.initials: PW
authors.lastname: King
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D007506:Iron-Sulfur Proteins; D006859:Hydrogen; C118830:iron hydrogenase; D006864:Hydrogenase