Scarrow R C, Brennan B A, Cummings J G, Jin H, Duong D J, Kindt J T, Nelson M J
Department of Chemistry, Haverford College, Pennsylvania 19041-1392, USA.
Biochemistry. 1996 Aug 6;35(31):10078-88. doi: 10.1021/bi960164l.
The iron K-edge X-ray absorption spectrum of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase in frozen solutions at pH 7 and 9 has been analyzed to determine details of the iron coordination. EXAFS analysis implies two or three sulfur ligands per iron and overall six coordination; together with previous EPR and ENDOR results, this implies an N3S2O ligation sphere. The bond lengths from EXAFS analysis [rav(Fe-S) = 2.21 A at pH 7.3; rav(Fe-N/O) = 1.99 A] support cis coordination of two cysteine ligands and conclusively rule out nitric oxide coordination to the iron, a possibility proposed on the basis of an FTIR difference experiment [Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I. (1995) FEBS Lett. 358, 9-12]. The higher-frequency EXAFS can be simulated well by inclusion of multiple scattering from two or three imidazole ligands; the fit to the data is improved if first-sphere multiple scattering pathways are also included. A slight shortening (by 0.02 +/- 0.01 A) of one or both Fe-S bonds when the pH is raised from 7.3 to 9.0 is consistent with shifts observed in the Raman spectrum [Brennan et al. (1996) Biochemistry 35, 10068-10077].
已对红球菌属R312（原短杆菌属R312）腈水合酶在pH值为7和9的冷冻溶液中的铁K边X射线吸收光谱进行了分析，以确定铁配位的细节。扩展X射线吸收精细结构（EXAFS）分析表明每个铁有两个或三个硫配体，且总体为六配位；结合之前的电子顺磁共振（EPR）和电子核双共振（ENDOR）结果，这意味着存在一个N3S2O配位球。EXAFS分析得到的键长[在pH 7.3时，rav(Fe - S)=2.21 Å；rav(Fe - N/O)=1.99 Å]支持两个半胱氨酸配体的顺式配位，并最终排除了一氧化氮与铁配位的可能性，而基于傅里叶变换红外光谱（FTIR）差异实验曾提出过这种可能性[Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I. (1995) FEBS Lett. 358, 9 - 12]。通过包含来自两个或三个咪唑配体的多重散射，可以很好地模拟高频EXAFS；如果也包括第一配位层的多重散射路径，则对数据的拟合会得到改善。当pH值从7.3升高到9.0时，一个或两个Fe - S键略有缩短（缩短0.02±0.01 Å），这与拉曼光谱中观察到的位移一致[Brennan等人(1996) Biochemistry 35, 10068 - 10077]。
