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 perform a variety of dioxygen-dependent essential functions, including the biosynthesis of DNA (ribonucleotide reductase), iron storage (ferritin), and the hydroxylation of organic substrates (methane monooxygenase, arene hydroxylases, deoxyhypusine hydroxylase). In general, dioxygen activation is proposed to entail a common mechanism involving diiron(III)- peroxo intermediates. Important project goals are to understand how the diiron(III)-peroxo intermediates are converted to corresponding high-valent iron-oxo species that very likely 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. The biochemical effort will primarily focus on human deoxyhypusine hydroxylase, an 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. This enzyme has a diiron active site and is isolated in an unusually stable diiron(III)-peroxo form that is nevertheless capable of substrate hydroxylation. The diiron active site will be investigated by a combination of biochemical and spectroscopic techniques to gain insight into its mode of action. The 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. Of particular interest are complexes with unusual Fe(III)-O-Fe(IV) and Fe(IV)-O-Fe(IV) units, motifs associated with the oxidizing species formed during enzyme catalysis. These novel complexes will be characterized by 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 (RNR) 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. 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 chlamydiae.

Public Health Relevance

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 chlamydiae.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM038767-24
Application #
8101490
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1999-04-01
Project End
2015-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
24
Fiscal Year
2011
Total Cost
$306,139
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Komor, Anna J; Rivard, Brent S; Fan, Ruixi et al. (2017) CmlI N-Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates. Biochemistry 56:4940-4950
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Komor, Anna J; Rivard, Brent S; Fan, Ruixi et al. (2016) Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI. J Am Chem Soc 138:7411-21
Jasniewski, Andrew J; Engstrom, Lisa M; Vu, Van V et al. (2016) X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a. J Biol Inorg Chem 21:605-18
Jasniewski, Andrew J; Knoot, Cory J; Lipscomb, John D et al. (2016) A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme ?-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase. Biochemistry 55:5818-5831
Ye, Shengfa; Xue, Genqiang; Krivokapic, Itana et al. (2015) Magnetic circular dichroism and computational study of mononuclear and dinuclear iron(IV) complexes. Chem Sci 6:2909-2921

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