This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR007707-16A1
Application #
7726019
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (40))
Project Start
2008-09-01
Project End
2009-07-31
Budget Start
2008-09-01
Budget End
2009-07-31
Support Year
16
Fiscal Year
2008
Total Cost
$7,908
Indirect Cost
Name
University of Chicago
Department
Miscellaneous
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
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