This program is devoted to solving central questions in metallobiochemistry through the use of multinuclear, multifrequency (9, 35, 95 GHz) CW and pulsed electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize transition metal centers that are vital to human health and disease. With these techniques we are able to completely characterize the active-site environment of a biological metal ion, and do so for key catalytic intermediates trapped by rapid freeze-quench and cryoreduction methods. When combined with isotopic labeling enabled by methods of modern molecular biology, these techniques permit the use of ENDOR/ESEEM to structurally characterize every stage of an enzyme's catalytic cycle. The current grant period further has shown that novel kinetic protocols can augment such structural information about intermediates with fundamental information about their reactivity. Together, these techniques provide the means to determine in precise detail how metal centers function in biology.
The specific aims of this program have expanded to include: formation of bioavailable nitrogen through enzymatic reduction of N2;anabolic and catabolic activation of O2 by heme and nonheme Fe enzymes;physiological protection against oxidative stress and contrasting toxicity, afforded by 'inorganic'Mn2+;enzymatic control of radical reactions in the 'Radical SAM'(S-adenosyl methionine) enzyme superfamily;development of new ENDOR methodologies and new applications in biology (Table 1). Table 1:
Aims for the Coming Period. N2 Reduction by Nitrogenase: Intermediates and Mechanism Biomimetic Mo and Fe Complexes in vivo EPR/ENDOR of Mn2+: Protection from Oxidative Stress Mechanisms of Toxicity Dioxygen-activation by Fe enzymes: Heme Monooxygenases Nonheme Fe Enzymes Radical Reactions in Metalloenzymes:The """"""""Radical SAM"""""""" Superfamily 'ENDOR Development: Hyperfine Signs Q-band Tubes at W Band.

Public Health Relevance

Metal centers play a central role in human health and disease. Our program is devoted to the development and application of advanced paramagnetic resonance measurement techniques as a uniquely incisivemeansofrevealinghowbiologicallyimportantmetalcentersfunction.Thesystemstobestudiedplay manyessentialroles,including:protectionfromoxidativestressandreactive-oxygenspecies;formationofthe key signaling and immune-response molecule, nitric oxide;xenobiotic and drug metabolism;antibiotic formation and resistance;regulation of diabetes-related metabolic pathways;enzymatic control of metabolically vital free-radical reactions;enzymatic formation of the bio-available nitrogen on which two- thirds of the world's population depend.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Goldsmith, Jonathan C
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Northwestern University at Chicago
Schools of Arts and Sciences
United States
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Davydov, Roman; Im, Sangchoul; Shanmugam, Muralidharan et al. (2016) Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy. Biochemistry 55:869-83
Shen, Jingmei; Kung, Mayfair C; Shen, Zhongliang et al. (2014) Generating and stabilizing Co(I) in a nanocage environment. J Am Chem Soc 136:5185-8
Davydov, Roman; Labby, Kristin Jansen; Chobot, Sarah E et al. (2014) Enzymatic and cryoreduction EPR studies of the hydroxylation of methylated N(?)-hydroxy-L-arginine analogues by nitric oxide synthase from Geobacillus stearothermophilus. Biochemistry 53:6511-9
Ortony, Julia H; Newcomb, Christina J; Matson, John B et al. (2014) Internal dynamics of a supramolecular nanofibre. Nat Mater 13:812-6
Hoffman, Brian M; Lukoyanov, Dmitriy; Yang, Zhi-Yong et al. (2014) Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem Rev 114:4041-62
Lukoyanov, Dmitriy; Yang, Zhi-Yong; Duval, Simon et al. (2014) A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates. Inorg Chem 53:3688-93
Hoffman, Brian M; Lukoyanov, Dmitriy; Dean, Dennis R et al. (2013) Nitrogenase: a draft mechanism. Acc Chem Res 46:587-95
Sharma, Ajay; Gaidamakova, Elena K; Matrosova, Vera Y et al. (2013) Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to ?-radiation by advanced paramagnetic resonance methods. Proc Natl Acad Sci U S A 110:5945-50
Davydov, Roman; Dawson, John H; Perera, Roshan et al. (2013) The use of deuterated camphor as a substrate in (1)H ENDOR studies of hydroxylation by cryoreduced oxy P450cam provides new evidence of the involvement of compound I. Biochemistry 52:667-71
Yang, Zhi-Yong; Khadka, Nimesh; Lukoyanov, Dmitriy et al. (2013) On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase. Proc Natl Acad Sci U S A 110:16327-32

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