The overall goal of this proposal is to understand how dioxygen is activated by biological diiron centers in metabolically critical transformations. Nonheme diiron enzymes perform a variety of essential functions involving dioxygen, including DNA biosynthesis (ribonucleotide reductase), iron storage (ferritin), and oxidations of organic substrates (methane monooxygenase, fatty acid desaturases, alkane and arene hydroxylases, myo-inositol oxygenase, deoxyhypusine hydroxylase). In general, dioxygen activation is proposed to entail a common mechanism involving diiron(III)-peroxo intermediates and high-valent iron-oxo species derived therefrom. The project goals will be accomplished using a combination of biomimetic and spectroscopic approaches. Building on past accomplishments in modeling structural and spectroscopic properties of such sites, it is proposed to synthesize precursor complexes of tripodal ligands, to react them with O2 or peroxides, and to characterize the metastable intermediates derived therefrom. Of great interest are intermediates such as O2 adducts of diiron(II) complexes (either iron(II)iron(III)-superoxo or diiron(III)-peroxo species), and species with Fe(III)Fe(IV) and Fe(IV)Fe(IV) oxidation states. These complexes will be characterized by X-ray crystallography whenever possible and by a variety of techniques such as NMR, EPR, UV-vis-NIR, Raman, M""""""""ssbauer, electrospray mass spectrometry, electrochemistry, and EXAFS. Parallel to these efforts, our spectroscopic expertise will be applied to elucidating the diiron site structures of methane monooxygenase intermediates and human deoxyhypusine hydroxylase. In this competitive revision, a new specific aim for GM-38767 is added because of the recent discovery that ribonucleotide reductase (RNR) of the parasite Chlamydia trachomatis uses as the oxidant needed to initiate ribonucleotide reduction a Fe(III)-O-Mn(IV) center, rather than the diiron(III)/tyrosyl radical combination characterized in E. coli and mammalian RNRs. It is suggested that this metal substitution allows this parasitic bacterium to circumvent the sensitivity of diiron RNR to NO produced in the typical immune response of mammalian cells. For the new specific aim, it is proposed to use methodologies developed in our ongoing work on synthetic diiron intermediates to obtain and characterize corresponding FeMn analogs, namely Fe(III)Mn(III)-peroxo, Fe(III)-O-Mn(IV), and Fe(IV)-O-Mn(IV) species. The properties of these novel FeMn complexes will be compared with those of their diiron counterparts to assess the chemical basis for Nature's choice of metal centers in this crucial enzyme.
Nonheme diiron enzymes perform a variety of metabolically critical functions that require dioxygen activation. Understanding how these enzymes work can lead to the development of new drug strategies for treating some human diseases. For example, ribonucleotide reductase is a key enzyme that controls DNA biosynthesis, while deoxyhypusine hydroxylase is required for the formation of mature eukaryotic elongation factor 5a that is essential for cell proliferation;thus both enzymes may serve as targets for anti-tumor or anti-HIV therapy.
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