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

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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 (