The structure of the Ni–Fe site in the isolated HoxC subunit of the hydrogen-sensing hydrogenase from Ralstonia eutropha

The regulatory Ni–Fe hydrogenase (RH) from Ralstonia eutropha which forms a [HoxBC]2 complex functions as a hydrogen sensor under aerobic conditions. We have studied a novel Strep-tag isolate of the RH large subunit, HoxCST, which lacks the Fe–S clusters of HoxB, allowing for structure determination of the catalytic site by X-ray absorption spectroscopy both at the Ni and, for the first time, also at the Fe K-edge. This technique, together with Fourier-transform infrared spectroscopy, revealed a Ni–Fe site with [O1(CysS)2NiII(μ-SCys)2FeII(CN)2(CO)] structure in about 50% of HoxCST and a [(CysS)2FeII(CN)2(CO)] site lacking Ni in the remainder protein. Possibly both sites may be intermediates in the maturation process of the RH.