Single crystal EPR studies of the reduced active site of [NiFe] hydrogenase from Desulfiovibrio vulgaris Miyazaki F
Single crystal EPR studies of the reduced active site of [NiFe] hydrogenase from Desulfiovibrio vulgaris Miyazaki F
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DOI：
10.1021/ja027522u
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发表时间：
2003-01-08
影响因子：
15
通讯作者：
Lubitz, W
中科院分区：
文献类型：
--
作者：
Foerster, S;Stein, M;Lubitz, W
在[NiFe]氢化酶的催化循环中，顺磁性Ni-C中间体至关重要，因为它被认为携带底物氢，尽管其几何形状尚不清楚。在低温照射下，Ni-C 转化为所谓的 Ni-L 态，具有明显不同的光谱参数。怀疑Ni-L失去了“底物氢”。在这项工作中，两种顺磁态均在从 Desulfovibrio, vulgaris Miyazaki F 的 [NiFe] 氢化酶获得的单晶中产生。对取向相关光谱的评估得出了 Ni-C 和 Ni-L 的 g 张量及其在晶轴系统中的取向。通过与还原酶的 X 射线晶体结构进行比较，g 张量可以进一步与原子结构相关。尽管 Ni-C 和 Ni-L 的 g 张量大小差异很大，但所得 g 张量的方向非常相似，但与之前获得的 Ni-A 和 Ni-B 不同 (Trofanchuk et al. J Biol. Inorg. Chem. 2000, 5, 36-44)。还使用活性位点的各种结构模型通过密度泛函理论（DFT）方法计算了 g 张量。计算出的 Ni-C g 张量在大小和方向方面与 Ni 和 Fe 原子之间具有氢化物 (H-) 桥的正式 Ni(III) 氧化态的实验结果非常一致。当假定该物质处于正式的 Ni(l) 氧化态并且从镍和铁原子之间的桥上除去质子 (HI) 时，Ni-L 状态获得了令人满意的一致性。
In the catalytic cycle of [NiFe] hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] hydrogenase from Desulfovibrio, vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the g tensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H-) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(l) oxidation state is assumed for this species with a proton (HI) removed from the bridge between the nickel and the iron atom.