Haumann Michael, Porthun Antje, Buhrke Thorsten, Liebisch Peter, Meyer-Klaucke Wolfram, Friedrich Bärbel, Dau Holger
Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany.
Biochemistry. 2003 Sep 23;42(37):11004-15. doi: 10.1021/bi034804d.
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, RH(ox)) 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 RH(ox) and RH(+H2) that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H(2) are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RH(ox)) to 8341.1 eV (RH(+H2)) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H(2). 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 Ni(II)(O,N)(2)S(3) for RH(ox) and six-coordinated Ni((III))(O,N)(3)X(1)S(2) [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.
首次通过镍K边X射线吸收光谱（XAS）对真养产碱菌氢传感器的镍位点——调节型[NiFe]氢化酶（RH）进行了研究。在不存在氢气（空气氧化，RH(ox)）和存在氢气（RH(+H2)）的情况下，对RH中镍位点的氧化态和原子结构进行了研究。发现与氢气孵育会导致光谱性质发生显著变化。仅在RH(+H2)状态下可检测到指示Ni(III)的Ni-C EPR信号。X射线吸收近边结构（XANES）和扩展X射线吸收精细结构（EXAFS）光谱表明，RH(ox)和RH(+H2)中镍的配位与标准[NiFe]氢化酶中的配位明显不同。此外，暴露于H2引起的变化是独特的。观察到XANES光谱发生剧烈变化，K边能量从8339.8（RH(ox)）上移至8341.1 eV（RH(+H2)）。EXAFS光谱表明，暴露于H2后，RH中镍的配位发生了变化。对数据的一种可能解释是，RH(+H2)中一个硫配体脱离，额外的（O，N）或H配体结合。提出了以下镍氧化态和配位：RH(ox)为五配位Ni(II)(O,N)(2)S(3)，RH(+H2)为六配位Ni((III))(O,N)(3)X(1)S(2) [X为（O，N）或H配体]。讨论了RH镍位点的结构特征与其功能——氢传感之间的关系。
