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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 为了对血红素-铜氧化酶的生物化学有更多的新认识，需要用连续的X射线束时间对嗜热链霉菌的新型细胞色素c氧化酶Ba3进行结晶学研究。我们的计划要求获得：(A)提高分离的、完全氧化的酶的新的突变形式的分辨率；(B)大量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的结构，以期在不同的氧化和连接状态下提高结晶的可靠性，我们希望通过合理的筛选，将提供~2.0A的分辨率，(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，然后在允许形成低温稳定的A-化合物Fea3-O2的条件下，使用低温流动系统与气体O2进行原位氧化。这构成了我们努力的主要目标。然而，我们仍然对Thermus Rieske蛋白感兴趣。以前的研究表明，在pH 8.5时，完全氧化的Thermus Rieske蛋白具有1.3°结构，其中一个铁结合的咪唑环是部分去质子化的。我们的计划要求将这项工作扩大到包括以下内容：(A)原子决议(

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 (