The diiron-oxo proteins have active sites consisting of metal centers bridged by oxo or hydroxo groups supported by carboxylate bridges. This expanding class of metalloproteins now includes proteins that perform a variety of functions in biology: dioxygen transport (hemerythrin), the conversion of ribonucleotides to deoxyribonucleotides (ribonucleotide reductase), iron storage (ferritin), phosphate ester hydrolysis (purple acid phosphatases), and oxidations of organic substrates via oxygen activation (methane monooxygenase, fatty acid desaturases, alkane and arene hydroxylases). Both soluble and membrane-bound forms are known. Many of the soluble enzymes have a sequence motif indicative of a carboxylate-rich diiron site, while the emerging membrane-bound subclass appears to have a histidine-rich diiron site. The focus of this proposal is to understand oxygen activation by diiron centers using a combination of biomimetic and biophysical approaches. Oxygen activation at a diiron active site is proposed to entail a common mechanism involving diiron(III)-peroxo intermediates and high-valent iron-oxo species derived therefrom. Building on past accomplishments in modeling structural and spectroscopic properties of such sites, it is proposed to synthesize precursor complexes that react with O2 or peroxides to afford metastable intermediates and characterize the spectroscopic and reactivity properties of these intermediates. Of great interest are intermediates such as O2 adducts of diiron(II) complexes, peroxo derivatives of iron(III), and species with Fe(III)Fe(IV) and Fe(IV)Fe(IV) formal oxidation states. These complexes will be characterized by x-ray crystallography whenever possible and by a variety of spectroscopic techniques such as NMR, EPR, UV-vis-NIR, Raman, Mossbauer, electrospray mass spectrometry, and EXAFS. Both stopped-flow and conventional kinetic methods will be used to characterize the mechanisms of formation and decomposition. The oxidative reactivities of these transient complexes towards a range of substrates will be investigated and compared with those of enzyme active sites. In parallel, EXAFS and resonance Raman studies of nonheme diiron enzyme intermediates themselves will be carried out to gain insight into their core structures. Of specific interest are the peroxo intermediates of stearoyl-ACP delta-9-desaturase and human H chain and E. coli ferritins as well as intermediates O, P, Q, and T in the methane monooxygenase cycle. These biophysical experiments dovetail well with the efforts to generate synthetic models for these enzyme intermediates.
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