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

摘要

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 H-2. Among them, the well-known H-ox state and the recently identified H-hyd state are thought to be directly involved in H-2 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 C-13 into the CO or CN- ligands and Fe-57 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 C-13 hyperfine couplings ((CO)-C-13 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 Fe-57 hyperfine couplings (Fe-57 a(iso) similar to 17of and 5.7 MHz) arising from the two Fe atoms in the [2Fe](H) subcluster. For H-hyd, we only observed two distinct (CO)-C-13 hyperfine interactions ((CO)-C-13 a(iso) of 0.16 and 0.08 MHz) and only one for (CN-)-C-13 ((CN)-C-13 a(iso) of 0.16 MHz); the couplings to the (CO)-C-13/(CN-)-C-13 on the distal Fe of [2Fe](H) may be too small to detect. We also observed a very small (0.3 MHz) Fe-57 HFI from the labeled [2Fe](H) subcluster and four Fe-57 HFI from the labeled [4Fe-4S](H) subcluster (Fe-57 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.