An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase
An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase
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DOI：
10.1007/s00775-004-0613-5
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发表时间：
2005-01-01
影响因子：
3
通讯作者：
Lubitz, W
中科院分区：
文献类型：
--
作者：
Foerster, S;van Gastel, M;Lubitz, W
应用电子核双共振（ENDOR）和超精细次能级相关谱（HYSCORE）研究了普通脱硫弧菌（Desulfovibrio vulgaris）宫崎F）[NiFe]-氢化酶在还原态Ni-C中的活性中心.这些技术提供了一个强大的工具，用于检测附近的磁性核，包括金属结合的衬底氢，并映射在活性位点的未成对电子的自旋密度分布。通过与来自X射线晶体学的结构数据和Ni-C状态中的完整g张量的知识进行比较来指定观察到的超精细耦合（Foerster等人（2003）J Am Chem Soc 125：83-93）。这被发现是在良好的协议与密度泛函理论计算。两个最强耦合的质子（a（iso）=13.7，11.8MHz）被指定为镍配位半胱氨酸549的β-CH 2质子，第三个质子（a（iso）= 8.9MHz）被指定为半胱氨酸546的β-CH 2质子。使用D2 O交换实验，氢化物在镍和铁之间的桥接位置的存在-最近在调节氢化酶中被检测到（布雷希特等人（2003）J Am Chem Soc 125：13075-13083）-首次在催化氢化酶中被实验证实。氢化物表现出小的各向同性超精细耦合常数（a（iso）=-3.5MHz），因为它在垂直于Ni（3d（z）2）轨道的z轴的方向上与Ni结合。已经鉴定了属于His-88的氮N-末端的氮信号。该残基与Cys-549的自旋携带Ni配位的硫形成氢键。与其他氢化酶的比较表明，活性位点在所有蛋白质中基本相同，包括调节性氢化酶。
Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83-93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons (a(iso)=13.7, 11.8 MHz) are assigned to the beta-CH2 protons of the nickel-coordinating cysteine 549, and a third proton (a(iso) = 8.9 MHz) is assigned to a beta-CH2 proton of cysteine 546. Using D2O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron - recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075-13083) - is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant (a(iso) = -3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni (3d(z)2) orbital. Nitrogen signals that belong to the nitrogen N-epsilon of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase.