Brecht Marc, van Gastel Maurice, Buhrke Thorsten, Friedrich Bärbel, Lubitz Wolfgang
Max-Volmer-Laboratorium für Biophysikalische Chemie, Fakultät für Mathematik und Naturwissenschaften, Technische Universität Berlin, D-10623 Berlin, Germany.
J Am Chem Soc. 2003 Oct 29;125(43):13075-83. doi: 10.1021/ja036624x.
The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.
β-变形菌罗尔斯通氏真养菌的调节型H2感应[NiFe]氢化酶在用H2还原后呈现出一种Ni-C“活性”状态，该状态与标准[NiFe]氢化酶的还原态Ni-C状态非常相似。脉冲电子核双共振（ENDOR）和四脉冲电子自旋回波包络调制（ESEEM，超精细能级相关，HYSCORE）光谱法被用于通过检测电子-核超精细耦合常数来获取该状态的结构信息。通过分析在EPR谱的整个磁场范围内记录的ENDOR谱，确定了两个质子超精细耦合。这些耦合与不可交换质子相关，属于NiFe中心桥连半胱氨酸的β-CH(2)质子。第三个质子的信号表现出很大的各向异性耦合（Ax = 18.4 MHz，Ay = -10.8 MHz，Az = -18 MHz）。在用2H2O代替H2O进行交换并用2H2而不是H2气体激活后，它们从ENDOR谱的1H区域消失。它们在ENDOR和HYSCORE谱的2H区域重新出现。基于与光谱相似的普通脱硫弧菌宫崎F型[NiFe]氢化酶的比较（已知其Ni-C状态的g张量相对于晶体结构的取向，Foerster等人，《美国化学会志》2003年，125卷，83 - 93页），提出了1H超精细耦合的归属。可交换质子位于Ni和Fe之间的桥连位置，被指定为一个形式上的氢负离子。在低温（T = 10 K）下光照后，形成了Ni-L状态。对于Ni-L状态，在Ni-C中可交换氢所观察到的强超精细耦合消失，表明金属-氢键发生了断裂。这些实验首次给出了关于调节型氢化酶活性NiFe中心中氢结合位置的直接信息。有人提出，在标准氢化酶的活性Ni-C状态中也存在这样的结合情况。
