Technische Universität Berlin, Strabetae des 17. Juni 135, 10623 Berlin.
Phys Chem Chem Phys. 2010 Mar 7;12(9):2139-48. doi: 10.1039/b922236g. Epub 2010 Jan 15.
The geometric and electronic structures of the active sites in the oxidized Ni(r)-B state of the [NiFe] hydrogenases from Ralstonia eutropha H16 and Desulfovibrio vulgaris Miyazaki F were investigated in pulsed EPR and ENDOR experiments at two different microwave frequencies (X- and Q-band). Two hyperfine-couplings were clearly resolved in the frozen solution spectra arising from the beta-protons of the nickel-coordinating cysteine residues Cys549 and Cys586 from the Desulfovibrio vulgaris and Ralstonia eutropha hydrogenase, respectively. ESEEM spectroscopic experiments reveal the presence of a histidine in the second coordination sphere of the Ni. The spectroscopic data indicate that the electronic structures of the [NiFe] centers in both hydrogenases are identical in the Ni(r)-B state. However, additional spin couplings of the active site to further paramagnetic centers were identified for the Ralstonia eutropha hydrogenase. The respective couplings could be clearly resolved and simulated. The results from this study are discussed in view of the exceptional O(2)-tolerance of the Ralstonia hydrogenase.
氧化态 Ni(r)-B 的 [NiFe] 氢化酶中活性位点的几何和电子结构在脉冲 EPR 和 ENDOR 实验中进行了研究，实验使用了两个不同的微波频率（X-和 Q-波段）。来自脱硫弧菌和氧化亚铁硫杆菌的氢化酶中，镍配位半胱氨酸残基 Cys549 和 Cys586 的β-质子分别在冻结溶液光谱中清晰地分辨出两个超精细耦合。ESEEM 光谱实验表明 Ni 的第二配位球中存在组氨酸。光谱数据表明，两种氢化酶中[NiFe]中心在 Ni(r)-B 态的电子结构相同。然而，进一步鉴定了氧化亚铁硫杆菌氢化酶活性位点与其他顺磁中心的附加自旋偶合。可以清楚地分辨和模拟各自的偶合。本研究的结果考虑了氧化亚铁硫杆菌氢化酶对 O(2)的异常耐受性进行了讨论。
