Bleijlevens B, Faber B W, Albracht S P
Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.
J Biol Inorg Chem. 2001 Oct;6(8):763-9. doi: 10.1007/s007750100252.
In this study we report on thus-far unobserved proton hyperfine couplings in the well-known EPR signals of [NiFe] hydrogenases. The preparation of the enzyme in several highly homogeneous states allowed us to carefully re-examine the Ni(u), Ni(r), Ni(a)-C* and Ni(a)-L* EPR signals which are present in most [NiFe] hydrogenases. At high resolution (modulation amplitude 0.57 G), clear indications for hyperfine interactions were observed in the g(z) line of the Ni(r)* EPR signal. The hyperfine pattern became more pronounced in 2H2O. Simulations of the spectra suggested the interaction of the Ni-based unpaired electron with two equivalent, non-exchangeable protons (A1,2=13.2 MHz) and one exchangeable proton (A3=6.6 MHz) in the Ni(r)* state. Interaction with an exchangeable proton could not be observed in the Ni(u)* EPR signal. The identity of the three protons is discussed and correlated to available ENDOR data. It is concluded that the NiFe centre in the Ni(r)* state contains a hydroxide ligand bound to the nickel, which is pointing towards the gas channel rather than to iron.
在本研究中，我们报告了在[NiFe]氢化酶著名的电子顺磁共振（EPR）信号中迄今未观察到的质子超精细耦合。将该酶制备成几种高度均一的状态，使我们能够仔细重新检查大多数[NiFe]氢化酶中存在的Ni(u)、Ni(r)、Ni(a)-C和Ni(a)-L EPR信号。在高分辨率（调制幅度0.57 G）下，在Ni(r)* EPR信号的g(z)线中观察到了超精细相互作用的明确迹象。在2H2O中，超精细模式变得更加明显。光谱模拟表明，在Ni(r)状态下，基于镍的未成对电子与两个等效的、不可交换的质子（A1,2 = 13.2 MHz）和一个可交换的质子（A3 = 6.6 MHz）相互作用。在Ni(u) EPR信号中未观察到与可交换质子的相互作用。讨论了这三个质子的身份，并将其与可用的电子核双共振（ENDOR）数据相关联。得出的结论是，Ni(r)*状态下的NiFe中心包含一个与镍结合的氢氧根配体，该配体指向气体通道而非铁。
