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

通过 X 射线吸收光谱确定胆色素原合成酶的 ZN(II) 连接

• 批准号: 6205757
• 负责人: ROBERT C SCARROW
• 金额: --
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6205757
• 关键词: biological products; biomedical resource; model design /development; proteins; spectrometry

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.

锌（II）取代的细胞色素c（Zn-Cytc）的结构， 最近通过NMR解析，表明Zn处于一种不寻常的 与六个配体的配位（Anni等人，生物化学1995，34， 5744-5753）。 此外，荧光谱线窄化光谱具有 显示Zn在蛋白质展开时变成五配位的。 一个 在Cytc中Zn六配位状态是独特的，因为没有 任何六配位锌蛋白的例子，并且没有 六配位的锌卟啉。 此外，特别 锌与Cytc中五个氮原子和一个硫原子配位 这是令人惊讶的，因为即使在锌有机化合物中， 六配位（428种晶体结构中的27%）， 5-Zn-（S）1型。 综合起来，这些发现表明， 蛋白质调节锌与其结合的偏好和强度 Cytc中的配体。 Zn边扩展X射线吸收精细结构 进行Zn-Cytc的1H（EXAFS）研究以确定 H中心金属环境，特别是Zn金属的距离 它的配体及其性质。 天然和胍变性 测量中性pH下的HZn-Cytc，以及相应的 HFe（III）-Cytc参考。 实验EXAFS数据集拟合到 H理论曲线（FEFF 6.01），使用新的细化程序 是被抓的。 该数据分析建立了以下协调 Cytc中的Zn Hsite：天然蛋白质中的六配位Zn H环境（Zn-Np= 2.052 A，Zn-Met 80（SD）= 2.34 A，Zn-His（NE）= 2.371 A） 将变性样品中的五配位Zn态混合 H（Zn-Np=2.10A，Zn-His（NE）=2.10-2.20A）。 Fe-cyt-c数据显示， 与晶体学数据一致。 变性分析 细胞色素C仍在继续