This proposal seeks support for a program of spectroscopic and mechanistic studies of heme iron enzymes. Three important goals will be pursued. First, a fundamental aspect of protein structure will be tested: the Fe-S bond is retained in all oxidation states of cytochrome P450 and chloroperoxidase but only in ferric states of thiolate-ligated myoglobin (Mb) and cytochrome c peroxidase (CCP) mutants. The factors that lead to loss of thiolate ligation may be the same as in the active sites of the heme proteins that naturally lose thiolate ligation upon reduction. Guided by molecular modeling, Mb and CCP double/triple thiolate-ligated mutants will be prepared with H-bond donor amino acids positioned to stabilize the thiolate ligand toward reduction and oxoferryl formation. Formation of thiolate-ligated oxoferryl adducts would provide simple models for these important states. Second, use of UV-visible/near-IR magnetic circular dichroism (MCD) spectroscopy will be extended for axial ligand identification in heme and chlorin iron proteins. Heme enzymes are ubiquitous biomolecules; the function of each is significantly influenced by its axial ligands. Axial ligand identification in a new heme protein is always one of the first lines of study. MCD spectroscopy has already found great application for this purpose, but there is the potential to significantly extend its utility. Toward this end, a large number of axial ligand adducts will be prepared that involve ligand combinations not previously scrutinized by MCD. Next, MCD will be used to address key coordination structure issues for cystathione beta synthase, heme oxygenase, soluble guanylyl cyclase and iron chlorin-containing systems. The third goal is to study the mechanism of molecular oxygen activation by nitric oxide synthase (NOS) and P450. Oxyferrous NOS, stabilized at low temperatures, will be used for the first time as the starting point to address specific mechanistic questions. With P450, the putative peroxyferric intermediate reported with the D251N mutant will be characterized. Finally, spectroscopic experiments on oxyferrous states of thiolate-ligated heme proteins and their one-electron reduced products will significantly increase our knowledge of these important, but poorly understood heme states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026730-16
Application #
6385366
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Ikeda, Richard A
Project Start
1979-07-01
Project End
2004-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
16
Fiscal Year
2001
Total Cost
$244,321
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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