This proposal seeks support for a program of spectroscopic, physical and mechanistic studies of secondary amine mono-oxygenase and a continued effort to develop the spectroscopic technique known as magnetic circular dichroism for the study of metalloporphyrin-containing enzymes. The study of secondary amine mono-oxygenase will be done in the context of our knowledge of the only other heme-containing mono-oxygenase, cytochrome P- 450, so as to test the relationship between structure and function for the heme iron mono-oxygenase class of enzymes. Efforts will continue to learn about the physical structure of SAMO and to study the electron transfer pathway within the intact protein. Purification of the heme-containing subunit will facilitate the direct study of the heme group. The nature of the heme environment of the enzyme will be examined, the identity of the proximal and distal heme ligands will be assigned in all physiologically relevant protein states, and the extent of exposure of the heme macrocycle within the active site will be probed. Rapid kinetics/freeze quench experiments are planned in order to establish the order of intermediates in the reaction cycle. Determining the source of oxygen incorporated into the product, probing the mechanism for free-radical character, examining non- physiological oxygen and electron donor interaction with SAMO, and measuring the extent of uncoupling and the products of any uncoupling of electron uptake from product formation are important objectives in our plan to elucidate the mechanism of action of SAMO. Demonstrating the utility of magnetic circular dichroism (MCD) spectroscopy in establishing the identity of axial ligands in heme iron and related iron chlorin proteins is the major goal of the spectroscopic portion of this research program. Efforts will be expanded to include studies of structure-dependent charge transfer transitions in the near-IR region (NIR MCD). These approaches will be used to study hemoprotein H-450, site- specific mutants of myoglobin and P-450, guanylate cyclase and E. coli complex II (succinate:ubiquinone oxidoreductase) with UV-Vis and NIR MCD. In addition, further attention will be focused on the unusual heme prosthetic group and spectroscopic properties of myeloperoxidase. Reconstitution of a chlorin into a proximal tyrosine-containing mutant of myoglobin will definitively establish whether tyrosine is the axial ligand in E. coli HPII catalase. Reconstitution of a chlorin into P-450 will reveal the spectral properties and reactivity of a thiolate-ligated iron chlorin. Finally, particular attention will be focused on the properties of the presumed Compound I and II intermediates in chlorin-reconstituted horseradish peroxidase in relationship to the chlorin-containing E. coli heme d oxidase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026730-13
Application #
2174779
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1979-07-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
13
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of South Carolina at Columbia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Modi, Anuja R; Dawson, John H (2015) Oxidizing intermediates in P450 catalysis: a case for multiple oxidants. Adv Exp Med Biol 851:63-81
Smith, Aaron D; Modi, Anuja R; Sun, Shengfang et al. (2015) Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa. Biochemistry 54:2601-12
Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P et al. (2015) Control of carotenoid biosynthesis through a heme-based cis-trans isomerase. Nat Chem Biol 11:598-605
Draganova, Elizabeth B; Akbas, Neval; Adrian, Seth A et al. (2015) Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment. Biochemistry 54:6598-609
Zhong, Fangfang; Lisi, George P; Collins, Daniel P et al. (2014) Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins. Proc Natl Acad Sci U S A 111:E306-15
Sun, Yuhan; Zeng, Weiqiao; Benabbas, Abdelkrim et al. (2013) Investigations of heme ligation and ligand switching in cytochromes p450 and p420. Biochemistry 52:5941-51
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
Molitor, Bastian; Stassen, Marc; Modi, Anuja et al. (2013) A heme-based redox sensor in the methanogenic archaeon Methanosarcina acetivorans. J Biol Chem 288:18458-72
Sun, Shengfang; Sono, Masanori; Dawson, John H (2013) Mono- and bis-phosphine-ligated H93G myoglobin: spectral models for ferrous-phosphine and ferrous-CO cytochrome P450. J Inorg Biochem 127:238-45
Owens, Cedric P; Du, Jing; Dawson, John H et al. (2012) Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake. Biochemistry 51:1518-31

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