The overall goal of this proposal is to understand how dioxygen is activated by biological diiron centers to carry out metabolically critical transformations. Nonheme diiron enzymes exhibit a remarkable range of dioxygen-dependent functions they perform, including the biosynthesis of DNA (ribonucleotide reductase), iron storage (ferritin), the hydroxylation of organic substrates (methane monooxygenase, toluene monooxygenase, deoxyhypusine hydroxylase), the biosynthesis of antibiotics (CmlA and CmlI), and the production of biodiesel (cyanobacterial aldehyde deformylating oxygenase). Important project goals are to understand how the diiron(III)-peroxo intermediates are converted to corresponding high-valent iron-oxo species that act as the key oxidants for substrate transformation and to describe the structural, electronic, and reactivity properties of the high-valent intermediates. These goals will be accomplished by both biochemical and biomimetic approaches. Our biochemical effort will focus on human deoxyhypusine hydroxylase (hDOHH), a diiron enzyme that hydroxylates a deoxyhypusine residue on eukaryotic initiation factor 5A to generate a mature form that is required for eukaryotic cell proliferation and implicated in HIV-1 transcription initiation; understanding how this enzyme works can lead to new strategies for treating cancer and AIDS. This enzyme is isolated in an unusually stable diiron(III)-peroxo form that is nevertheless catalytically competent. The diiron active site will be investigated by a combination of kinetic and spectroscopic techniques to gain insight into its mode of action. In addition, spectroscopic studies will be carried out on CmlA and CmlI, bacterial enzymes involved in chloramphenicol biosynthesis to understand how these enzymes work. Our biomimetic effort will focus on generating and trapping metastable species that relate to diiron(III)- peroxo and high-valent iron intermediates observed in the redox cycles of the nonheme diiron enzymes. Novel complexes with reactive Fe(III)-O-Fe(IV) and Fe(IV)-O-Fe(IV) units will be characterized with a variety of spectroscopic techniques to determine their structures and electronic properties. Corresponding complexes with Fe(III)-O-Mn(IV) and Fe(IV)-O-Mn(IV) units will also be synthesized to model high-valent intermediates associated with the recently discovered ribonucleotide reductase with a FeMn active site (instead of a diiron site) from the parasite Chlamydia trachomatis. Understanding the difference in the reactivity properties of high- valent FeFe and FeMn complexes may contribute to the development of better methods for treating infections from such human pathogens. Various synthetic diiron-O2 adducts will be also investigated to assess their ability to effect oxidative deformylation of aldehydes as a model for ADO, which requires a peroxo moiety that is nucleophilic in character in order to carry out this transformation.
Nonheme diiron enzymes perform a variety of metabolically critical functions that require O2. For example, ribonucleotide reductase is a key enzyme that controls DNA biosynthesis, while deoxyhypusine hydroxylase is required for the formation of mature eukaryotic initiation factor 5a that is essential for cell proliferation. Understanding how these enzymes work can lead to the development of new drug strategies for treating cancer, AIDS, and infections of human pathogens like Chlamydia.
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