HIGH-RESOLUTION CRYSTALLOGRAPHIC STUDIES OF TWO RESPIRATORY PROTEINS FROM T THE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 为了获得对血红素-铜氧化酶生物化学的强有力的新见解，需要连续的X射线束时间来对来自T.嗜热菌我们的计划要求获得：（a）分离的完全氧化的酶的新突变形式的分辨率增加;（B）许多突变形式的独立结构，特别是：（i）在不同空间群中结晶的突变蛋白质，目的是增加在不同氧化和连接状态下结晶的可靠性，并且我们希望通过合理筛选将提供~2.0 A分辨率，（ii）揭示O2传递通道的结构和功能，（iii）检查在所谓的质子运输的K-路径中突变的结构效应;（c）完全还原的酶的独立结构，随后在允许形成低温稳定的A-化合物的条件下使用低温流动系统用气态O2原位氧化，Fea3-O2。这是我们努力的主要目标。然而，我们仍然对Thermus Rieske蛋白感兴趣。先前的研究产生了在pH 8.5下完全氧化的Thermus Rieske蛋白的1.3 <$结构，其中一个Fe结合的咪唑环被推断为部分去质子化。我们的计划要求将这项工作扩展到包括以下内容：（a）原子分辨率（

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In order to garner powerful new insights on heme-Cu oxidase biochemistry, continued X-ray beam time is requested to perform crystallographic studies of the novel cytochrome c oxidase, ba3, from T. thermophilus. Our plans call for obtaining: (a) increased resolution of a new mutant form of the as-isolated, fully oxidized enzyme; (b) independent structures of numerous mutant forms notably: (i) of mutated proteins that crystallize in different space groups with the view of increasing reliability of crystallization in different oxidation and ligation states, and, we hope, through rational screening, will provide ~2.0 A resolution, (ii) to reveal both structure and function of the O2 delivery channel, (iii) to examine the structural effects of mutations in the so-called K-path of proton transport; (c) independent structures of the fully reduced enzyme followed by in situ oxygenation with gaseous O2 using a cryogenic flow system under conditions which permit formation of the cryo-stable A-compound, Fea3-O2. This constitutes the main objectives of our effort. However, we retain an interest in the Thermus Rieske protein. Previous studies yielded a 1.3 ¿ structure of the fully-oxidized Thermus Rieske protein at pH 8.5 in which one of the Fe-bound imidazole rings was deduced to be partially de-protonated. Our plans call for extending this work to include the following: (a) an atomic resolution (