Study of water binding to low-spin Fe(III) in cytochrome P450 by pulsed ENDOR and four-pulse ESEEM spectroscopies
Study of water binding to low-spin Fe(III) in cytochrome P450 by pulsed ENDOR and four-pulse ESEEM spectroscopies
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DOI：
10.1021/ja951307e
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发表时间：
1996-03-20
影响因子：
15
通讯作者：
Ullrich, V
中科院分区：
文献类型：
--
作者：
Goldfarb, D;Bernardo, M;Ullrich, V
用脉冲Endor和二、四脉冲ESEEM光谱仪研究了细胞色素P450 cam(CP450 Cam)。在菱形EPR谱的三个主g值处记录并模拟了光谱。四脉冲ESEEM实验给出了质子各向异性超精细相互作用的直接测量。使用点偶极子近似，得到的Fe-H距离为2.6埃。测量到的各向异性超精细相互作用减少了模拟Endor线形状所需的超精细相互作用参数的数量。三个主g值处的四脉冲ESEEM频率和Endor谱都可以用两个磁当量的质子和水的取向进行令人满意的模拟，因此，脉冲Endor和四脉冲ESEEM的结果与O-17 ESEEM的数据是自洽的，并表明轴向配体是水分子而不是OH-配体。由数值模拟得到的各向同性超精细值与质子核磁共振弛豫研究得到的值是一致的。
Cytochrome P450cam (CP450cam) was studied by pulsed ENDOR and two- and four-pulse ESEEM spectroscopies. Spectra were recorded and simulated at the three principal g-values of the rhombic EPR spectrum. The four-pulse ESEEM experiment gave a direct measure of the anisotropic hyperfine interaction for the protons. Using the point dipole approximation this gives a Fe-H distance of 2.6 Angstrom. The measured anisotropic hyperfine interaction reduced the number of hyperfine interaction parameters required to simulate the ENDOR line shapes. Both the four-pulse ESEEM frequencies and the ENDOR spectra at all three principal g-values could be satisfactorily simulated using two magnetically equivalent protons and a water orientation similar to that obtained in our previous O-17 ESEEM study Thus, the pulsed ENDOR and four-pulse ESEEM results are self-consistent with the O-17 ESEEM data and indicate that the axial ligand is a water molecule rather than an OH- ligand. The isotropic hyperfine value derived from the numerical simulations is in agreement with previous values derived from proton NMR relaxation studies.