Electronic and molecular structures of the active-site H-cluster in [FeFe]-hydrogenase determined by site-selective X-ray spectroscopy and quantum chemical calculations

The [FeFe]-hydrogenase (HydA1) from green algae is the minimal enzyme for efficient biological hydrogen (H-2) production. Its active-site six-iron center (H-cluster) consists of a cubane, [4Fe4S](H), cysteine-linked to a diiron site, [2Fe](H). We utilized the spin-polarization of the iron K beta X-ray fluorescence emission to perform site-selective X-ray absorption experiments for spectral discrimination of the two sub-complexes. For the H-cluster in reduced HydA1 protein, XANES and EXAFS spectra, K beta emission lines (3p -> 1s transitions), and core-to-valence (pre-edge) absorption (1s -> 3d) and valence-to-core (K beta(2,5)) emission (3d -> 1s) spectra were obtained, individually for [4Fe4S](H) and (2Fe)(H). Iron-ligand bond lengths and intermetal distances in [2Fe](H) and [4Fe4S](H) were resolved, as well as fine structure in the high-spin iron containing cubane. Density functional theory calculations reproduced the X-ray spectral features and assigned the molecular orbital configurations, emphasizing the asymmetric d-level degeneracy of the proximal (Fe-p) and distal (Fe-d) low-spin irons in [2Fe](H) in the non-paramagnetic state. This yielded a specific model structure of the H-cluster with a bridging carbon monoxide ligand and an apical open coordination site at Fe-d in [2Fe](H). The small HOMO-LUMO gap (similar to 0.3 eV) enables oxidation and reduction of the active site at similar potentials for reversible H-2 turnover by HydA1, the LUMO spread over [4Fe4S](H) supports its role as an electron transfer relay, and Fe-d carrying the HOMO is prepared for transient hydride binding. These features and the accessibility of Fe-d from the bulk phase can account for regio-specific redox transitions as well as H-2-formation and O-2-inhibition at the H-cluster. We provide a conceptual and experimental framework for site-selective studies on catalytic mechanisms in inhomogeneous materials.