EPR and FTIR Analysis of the Mechanism of H2 Activation by [FeFe]-Hydrogenase HydA1 from Chlamydomonas reinhardtii

• Published in: Journal of the American Chemical Society Vol. 135; no. 18; pp. 6921 - 6929
• Main Authors: Mulder, David W, Ratzloff, Michael W, Shepard, Eric M, Byer, Amanda S, Noone, Seth M, Peters, John W, Broderick, Joan B, King, Paul W
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08-05-2013
• Subjects: Chlamydomonas reinhardtii - enzymology; Electron Spin Resonance Spectroscopy; Enzyme Activation; Hydrogen - chemistry; Hydrogen - metabolism; Hydrogenase - chemistry; Hydrogenase - metabolism; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - metabolism; Models, Molecular; Molecular Conformation; Spectroscopy, Fourier Transform Infrared
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja4000257

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.