One major element of our program is the development of electron- nuclear double resonance (endor) spectroscopy as an essential tool in determining metalloenzyme structure and function. To this end we will develop Q-Band (35 GHz) endor as a standard measurement technique in metallobiochemistry because it will prevent proton- endor signals from obscuring the resonances of constitutive nuclei (14,15N, 13C, 17O, 57Fe, 33S) and will reduce requirements 10-20 fold, opening new prospects for studying scarce or isotopically labelled proteins. Q-Band, CW and pulsed-X Band, and double endor techniques will be applied to the study of a wide range of enzymes active sites, including the CuA and CuB sites of cytochrome oxidases, the atypical (2Fe, 2S) center of Rieske-type respiratory proteins and enzymes, and the multi-metal clusters of carbon monoxide dehydrogenase. In the second major element we employ metal-substituted hemoproteins to elucidate the factors regulating long-range electron transfer within protein-protein complexes. Mixed-metal, (M,M') hemoglobin hybrids, M = Zn,Mg, M' = Fe, Mn, Co, provide the ideal system for examining inter-protein electron transfer where the two redox centers are rigidly held within a structure that can be crystallographically determined to high precision. The complex cytochrome c peroxidase (CcP) and its physiological partner, cytochrome c (Cc), will be used to examine the role of the docking interface in regulating recognition and function, as well as the role of the protein matrix in modulating electron transfer. We shall use Cc from multiple species to explore the evolutionary matching of the interface with Zn and Mg-substituted CcP. Variants of CcP and of the Cc from yeast, drosophila, and rat that have been produced by site-specific mutagenesis will be used to explore the regulatory role of single amino acids and clusters both at the protein-protein interface and in the heme crevice. The complexes between cytochrome P450-cam with putadaredoxin and cytochrome b5 will provide a parallel system for study.
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