The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. I. Light sensitivity and magnetic hyperfine interactions as observed by electron paramagnetic resonance

The hydrogen-activating cluster (H cluster) in FeFe]-hydrogenases consists of two moieties. The 2Fe]_H subcluster is a (L)(CO)(CN)Fe(μ-RS₂)(μ-CO)Fe(CysS)(CO)(CN) centre. The Cys-bound Fe is called Fe1, the other iron Fe2. The Cys-thiol forms a bridge to a 4Fe–4S] cluster, the 4Fe–4S]_H subcluster. We report that electron paramagnetic resonance (EPR) spectra of the ⁵⁷Fe-enriched enzyme from Desulfovibrio desulfuricans in the H_ox–CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster. In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light. The 2Fe]_H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the H_ox–CO state giving rise to the so-called axial 2.06 EPR signal. Though not destroyed by light, the H_ox–CO state is affected by it. As demonstrated in the accompanying paper 49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic ¹³CO during illumination at 2 °C. We found that only one of the three ¹³COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 0.6 mT. At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed. EPR spectra of the photolysed products are consistent with a 3d ⁷ system of Fe with the formal oxidation state +1. The damaged enzyme shows a light-sensitive g=5 signal which is ascribed to an S=3/2 form of the 2Fe]_H subcluster. The light sensitivity of the enzyme explains the occurrence of the g=5 signal and the axial 2.06 signal in published EPR spectra of nearly all preparations studied thus far.