STRUCTURAL ANALYSIS OF THE MEMBRANE METALLOPROTEIN CYTOCHROME C OXIDASE IN

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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. Our proposed research involves x-ray crystallographic analysis of Cytochrome c oxidase (CcO) from Rhodobacter sphaeroides (Rs). CcO catalyzes the reaction vital in energy production by providing the final electron sink and reducing oxygen to water, while translocating protons across the membrane to form the electrochemical gradient used for direct ATP synthesis. Mechanistic studies of mitochondrial CcO are facilitated by studying its bacterial homologue, eg CcO from Rs. We recently obtained high resolution crystal structures of the I-II subunit catalytic core of the RsCcO in the oxidized form at 2.0 ¿ resolution, as well as the dithionite-reduced form of the enzyme at 2.2 ¿ resolution. In the reduced structure, an unusual displacement of heme a3 group was seen: the entire protoporphyrin ring as well as the hydroxyl-farnesyl tail is shifted and rotated slightly. As a result, the distance between the OH group of the hydroxyl-farnesyl tail and the OH group of Y288 becomes much greater than the usual tight hydrogen bonding distance (4.0 ¿ vs. 2.6 ¿), opening the top of the proton input channel (K path). The CuB ? heme a3 metal-metal distance is also greater (4.9 ¿ vs. 5.4 ¿) and density attributed to OH-/H2O at the active site in the oxidized form of the structure is gone. These changes were not seen in the published bovine heart mitochondrial CcO in the reduced form. Therefore, we need to confirm the redox states of the different forms of the crystals before, during and after X-ray data collection, especially given that the synchrotron radiation could generate photoelectrons to reduce oxidized crystals, and that oxygen could still be in contact with a reduced crystal, during data collection. The on-line microspectrophotometer available at BioCARS, 14-BM-C, is needed to conclusively determine the redox states of the crystal; the enzyme gives distinct spectral peaks in the visible spectrum region in the oxidized and reduced states.