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 H_ox state and the recently identified H_hyd 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 H_ox, we measured ¹³C hyperfine couplings (¹³CO a_iso 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 a_iso of ∼17 and 5.7 MHz) arising from the two Fe atoms in the [2Fe]_H subcluster. For H_hyd, we only observed two distinct ¹³CO hyperfine interactions (¹³CO a_iso of 0.16 and 0.08 MHz) and only one for ¹³CN^- (¹³CN a_iso 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 a_iso 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 H_ox and H_hyd states and provide a basis for more detailed analysis.