DETERMINE ZN(II) LIGATION OF PORPHOBILINOGEN SYNTHASE BY XRAY ABSORPTION SPEC

锌(II)取代的细胞色素c(锌-细胞色素c)的结构， 最近被核磁共振解析，表明锌在一种不寻常的 与六个配体的配位作用(Anni等人，生物化学1995，34， 5744-5753)。此外，荧光线窄化光谱还具有 结果表明，当蛋白质展开时，锌变成五配位。一个 锌在细胞色素C中的六配位状态是唯一的，因为没有 任何六配位的锌蛋白的例子，并且没有 溶液中的六配位锌卟啉。此外，特别的是 锌与细胞色素C中5个氮基和1个硫配体的配位作用 令人惊讶，因为即使在锌的有机化合物中 六配位的(428个晶体结构中的27%)都不是 类型(N)5-锌-(S)1。综合这些发现， 锌对其结合偏好和结合强度的蛋白质调节 Cytc中的配体。锌边扩展X射线吸收精细结构 对锌-细胞色素C进行了H(EXAFS)研究，以确定 中心金属环境，特别是锌金属的距离 它的配体和它们的性质。既有天然的，也有胍变性的 在中性pH下测定HZN-Cytc，以及相应的 HFe(III)-Cytc参考文献。实验的EXAFS数据集符合 用一种新的精化方法绘制的H理论曲线(FEFF 6.01) 都是他得到的。该数据分析建立了以下方面的协调 细胞色素C中的锌H位：天然蛋白质中的六配位锌 H环境(锌-NP=2.052A，锌-金属80(SD)=2.34A，锌-组氨酸(NE)=2.371A) 在变性样品中加入五配位锌的混合物 H(锌-NP=2.10A，锌-组氨酸(NE)=2.10-2.20A)。Fe-Cyt-c数据显示接近 与结晶学数据一致。变性剂的分析 细胞色素-c仍在继续。

The structure of zinc (II)-substituted cytochrome c (Zn-Cytc), recently resolved by NMR, suggested that Zn is in an unusual coordination with six ligands (Anni et al., Biochemistry 1995, 34, 5744-5753). Furthermore, fluorescence line narrowing spectroscopy has shown that Zn becomes penta-coordinated upon protein unfolding. An hexa-coordinated state of Zn in Cytc is unique since there are no examples of any hexa-coordinated Zn-proteins, and there are no hexa-coordinated Zn-porphyrins in solution. Moreover, the particular coordination of Zn with five nitrogens and one sulfur ligand in Cytc is surprising, because even among the Zn organic compounds that are hexa-coordinated (27% of the 428 crystal structures) none is of the type (N) 5-Zn-(S) 1. Taken together these findings point to a tight protein modulation of the binding preference and strength of Zn to its ligands in Cytc. The Zn-edge extended X-Ray absorption fine structure H(EXAFS) studies of Zn-Cytc were undertaken in order to define the Hcentral metal environment, specifically the distances of the Zn metal Hto its ligands and their nature. Both native and guanidine-denatured HZn-Cytc at neutral pH were measured, alongside with the corresponding HFe(III)-Cytc references. Sets of experimental EXAFS data were fit to Htheoretical curves (FEFF 6.01) using a novel refinement procedure were Hobtained. This data analysis establishes the coordination of the Zn Hsite in Cytc: an hexa-coordinated Zn in the native protein Henvironment (Zn-Np=2.052A, Zn-Met80 (SD) =2.34A, Zn-His(NE)=2.371A) Hand a mixture of penta-coordinated Zn states in the denatured sample H(Zn-Np=2.10A, Zn-His(NE) =2.10-2.20A). Fe-cyt-c data showed close Hagreement with crystallographic data. Analysis of denatured Hcytochrome-c is continuing.