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

Abstract

The [FeFe]-hydrogenase (HydA1) from green algae is the minimal enzyme for efficient biological hydrogen (H₂) 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 Kβ 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, Kβ emission lines (3p → 1s transitions), and core-to-valence (pre-edge) absorption (1s → 3d) and valence-to-core (Kβ^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 (∼0.3 eV) enables oxidation and reduction of the active site at similar potentials for reversible H₂ 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 H₂-formation and O₂-inhibition at the H-cluster. We provide a conceptual and experimental framework for site-selective studies on catalytic mechanisms in inhomogeneous materials.