Hydrogen-Induced Structural Changes at the Nickel Site of the Regulatory [NiFe] Hydrogenase from Ralstonia eutropha Detected by X-ray Absorption Spectroscopy

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Abstract
For the first time, the nickel site of the hydrogen sensor of Ralstonia eutropha, the regulatory [NiFe] hydrogenase (RH), was investigated by X-ray absorption spectroscopy (XAS) at the nickel K-edge. The oxidation state and the atomic structure of the Ni site were investigated in the RH in the absence (air-oxidized, RHox) and presence of hydrogen (RH+H2). Incubation with hydrogen is found to cause remarkable changes in the spectroscopic properties. The Ni-C EPR signal, indicative of Ni(III), is detectable only in the RH+H2 state. XANES and EXAFS spectra indicate a coordination of the Ni in the RHox and RH+H2 that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H2 are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RHox) to 8341.1 eV (RH+H2) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H2. One likely interpretation of the data is the detachment of one sulfur ligand in RH+H2 and the binding of additional (O,N) or H ligands. The following Ni oxidation states and coordinations are proposed: five-coordinated NiII(O,N)2S3 for RHox and six-coordinated Ni(III)(O,N)3X1S2 [X being either an (O,N) or H ligand] for RH+H2. Implications of the structural features of the Ni site of the RH in relation to its function, hydrogen sensing, are discussed.

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