The overall goal of this research is to understand how proteins that contain paramagnetic centers work. The applicant will probe the structure of the paramagnetic centers themselves, the interaction between the centers, and the correlation between the structure of the center and its protein surroundings. The systems chosen for study are a protein that recognizes and excises damages from DNA to eliminate potential mutations, proteins that couple transfer of electrons to production of biologically useful energy in mitochondria, and proteins and their models that catalyze biological oxidations. The applicant will focus on probing these centers primarily through the techniques of electron paramagnetic resonance (EPR) (pulsed and continuous) and electron nuclear double resonance (ENDOR) with significant use of other spectroscopic and kinetic methods. The applicant's laboratory is one of the few in the world which routinely uses ENDOR to measure hyperfine couplings of the electron spins in biological molecules. The specific systems to be studied are: (1) Endonuclease III of E. coli which is a DNA repair enzyme that contains, for reasons the applicant intends to discover, an iron-sulfur center. (2) Cytochrome c oxidase, which couples the transfer of electrons to molecular oxygen to the production of biologically useful energy. The applicant intends to probe the electronic mechanisms of this coupling at paramagnetic metals. (3) The mitochondrial cytochrome bc1 complex, which also couples the transfer of electrons through its paramagnetic centers to production of biologically useful energy. The applicant intends to probe the structure of free radicals and paramagnetic metals in the bc1 complex. (4) Cytochrome c peroxidase, which catalyzes the consumption of reduced cytochrome c by peroxide. In consuming peroxide, this enzyme gives rise to an unusual paramagnetic intermediate which the applicant will characterize by using magnetic resonance techniques on genetically manipulated protein. (5) Models for the active sites of copper proteins which catalyze oxidation of substrates or reversibly bind oxygen. As the model structure is varied, the applicant will study the resultant perturbations to electronic structure.
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