Electronic Structure of Two Catalytic States of the [FeFe] Hydrogenase H-Cluster As Probed by Pulse Electron Paramagnetic Resonance Spectroscopy.

The active site of the [FeFe] hydrogenase (HydA1), the H-cluster, is a 6-Fe cofactor that contains CO and CN- ligands. It undergoes several different oxidation and protonation state changes in its catalytic cycle to metabolize H2. Among them, the well-known Hox state and the recently identified Hhyd state are thought to be directly involved in H2 activation and evolution, and they are both EPR active with net spin S = 1/2. Herein, we report the pulse electronic paramagnetic spectroscopic investigation of these two catalytic states in Chlamydomonas reinhardtii HydA1 ( CrHydA1). Using an in vitro biosynthetic maturation approach, we site-specifically installed 13C into the CO or CN- ligands and 57Fe into the [2Fe]H subcluster of the H-cluster in order to measure the hyperfine couplings to these magnetic nuclei. For Hox, we measured 13C hyperfine couplings (13CO aiso of 25.5, 5.8, and 4.5 MHz) corresponding to all three CO ligands in the H-cluster. We also observed two 57Fe hyperfine couplings (57Fe aiso of ∼17 and 5.7 MHz) arising from the two Fe atoms in the [2Fe]H subcluster. For Hhyd, we only observed two distinct 13CO hyperfine interactions (13CO aiso of 0.16 and 0.08 MHz) and only one for 13CN- (13CN aiso of 0.16 MHz); the couplings to the 13CO/13CN- on the distal Fe of [2Fe]H may be too small to detect. We also observed a very small (<0.3 MHz) 57Fe HFI from the labeled [2Fe]H subcluster and four 57Fe HFI from the labeled [4Fe-4S]H subcluster (57Fe aiso of 7.2, 16.6, 28.2, and 35.3 MHz). These hyperfine coupling constants are consistent with the previously proposed electronic structure of the H-cluster at both Hox and Hhyd states and provide a basis for more detailed analysis.