Our overall goal is to understand how biological systems that contain paramagnetic centers work. We will probe the paramagnetic centers to determine local liganding structure, shared unpaired electron distribution, and evidence for protein-induced perturbation. We will investigate these centers primary through the techniques of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR). Besides EPR/ENDOR we will bring complementary kinetic and spectroscopic methods to bear as needed. The systems to be studied are: 1. Nitrite Reductase. This work aims to understand the catalytic mechanism of the Type 2 copper in nitrite reductase as it reduces nitrite to nitric oxide. 2. Fe-containing Bleomycin Interacting with DNA. The objective of this study will be to build upon our recent ENDOR study that showed the interaction of bleomycin iron with nearby DNA substrate. We will more completely delineate the proximity of bleomycin to DNA atoms and work toward understanding the identity of the axial nitrogenous ligand to the bleomycin iron. 3. MN++ Centers in DNA Repair Enzymes. The plan for this work is to probe the MN++-centered active sites of Endonuclease III and Exonuclease IV, two E. coli proteins which recognize DNA with damaged or lost bases and break the DNA phosphate backbone. Interaction of the MN++ with substrate and perturbation of the active site structure from mutations near the active site will be delineated by ENDOR. 4. Multimanganese Center for Photosynthesis. This work is aimed at probing the multimanganese center with high sensitivity Q-band ENDOR to obtain information on exchangeable 17/O-water and protons and that center and to understand the structural basis of the required Cl- and Ca++ in the function of the center. 5. ENDOR of the High Spin Ferric Heme Liganding Environment at the Oxygen- Consuming Center of Terminal Oxidase. The plan of this work is to uncover the liganding environment at and near the 6th heme ligand of the binuclear oxygen-consuming center of bo oxidase.
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