This proposal deals with experimental approaches to quantitative characterization of both the molecular and electronic structures of the active sites of moderate-sized (30-80 kDa) heme-containing enzymes in order to understand the mechanism (peroxidase vs. oxygenase) and stereospecificity (alpha vs. beta, gamma, or delta-meso heme cleavage) of the reactions with substrate. Solution NMR spectroscopy of the active site of heme enzymes in paramagnetic states is ideally suited for providing detailed description of such active sites, where advantage is taken of both the increased spectral dispersion that significantly facilitates resolution for active site residues, and the great wealth of novel structural information contained in the hyperfine shifts responsible for the shift dispersion. Methodological approaches will emphasize more quantitative structural studies (internuclear distance) of 30-40 kDa low-spin, ferric enzymes and extension of both assignments and structural analyses to larger 78 kDa) low-spin, ferric, and more strongly paramagnetic, 30-40 kDa high-spin, ferric heme enzymes. The methodological advances are expected to contribute not only to effective structural characterization of our own target enzymes, but to contribute significantly to the NMR investigation of a large variety of other paramagnetic metalloproteins currently pursued in other research laboratories. The optimally developed NMR strategies will be applied to determining in detail the effect of mutation and ligation on the nature of the heme cavity and substrate binding pocket of 44 kDa horseradish peroxidase, and elucidating the heme electronic structure that results from the covalent heme links to the protein matrix in 78 kDa lactoperoxidase. Studies on ligated and substrate-bound human heme oxygenase will focus on reconciling the apparent structural differences between the ligated complex in solution and the unligated complex in the crystal with respect to the position of the distal helix, the nature of the distal hydrogen-bond donor to the bound ligand, the identity of the titrating group that inactivates the protein, the exact origin of steric tilt of the ligand in the direction of the alpha-meso position, and the nature of the distal hydrogen-bonds between two Tyr that are likely involved in """"""""closing"""""""" the substrate cavity prior to reaction. The effect of mutation on these structural features will also be examined. The prospects for definitive solution NMR studies of cytochrome P450 will also be explored. It is expected that a considerable improved understanding of the solution structures of the target enzyme and the relationship to their functions will result from these studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM062830-01
Application #
6317416
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Wehrle, Janna P
Project Start
2001-05-01
Project End
2005-04-30
Budget Start
2001-05-01
Budget End
2002-04-30
Support Year
1
Fiscal Year
2001
Total Cost
$172,625
Indirect Cost
Name
University of California Davis
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Davis
State
CA
Country
United States
Zip Code
95618
Peng, Dungeng; Ogura, Hiroshi; Ma, Li-Hua et al. (2013) Solution NMR characterization of magnetic/electronic properties of azide and cyanide-inhibited substrate complexes of human heme oxygenase: implications for steric ligand tilt. J Inorg Biochem 121:179-86
Peng, Dungeng; Ma, Li-Hua; Smith, Kevin M et al. (2012) Role of propionates in substrate binding to heme oxygenase from Neisseria meningitidis: a nuclear magnetic resonance study. Biochemistry 51:7054-63
Peng, Dungeng; Satterlee, James D; Ma, Li-Hua et al. (2011) Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis. A 1H NMR study. Biochemistry 50:8823-33
Peng, Dungeng; Ma, Li-Hua; Ogura, Hiroshi et al. (2010) 1H NMR study of the influence of mutation on the interaction of the C-terminus with the active site in heme oxygenase from Neisseria meningitidis: implications for product release. Biochemistry 49:5832-40
Du, Zhenming; Unno, Masaki; Matsui, Toshitaka et al. (2010) Solution 1H NMR characterization of substrate-free C. diphtheriae heme oxygenase: pertinence for determining magnetic axes in paramagnetic substrate complexes. J Inorg Biochem 104:1063-70
Ogura, Hiroshi; Evans, John P; Peng, Dungeng et al. (2009) The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: implications for the enzyme mechanism. Biochemistry 48:3127-37
Ma, Li-Hua; Liu, Yangzhong; Zhang, Xuhong et al. (2009) 1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure. J Inorg Biochem 103:10-9
Peng, Dungeng; Ogura, Hiroshi; Zhu, Wenfeng et al. (2009) Coupling of the distal hydrogen bond network to the exogenous ligand in substrate-bound, resting state human heme oxygenase. Biochemistry 48:11231-42
Ogura, Hiroshi; Evans, John P; de Montellano, Paul R Ortiz et al. (2008) Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation. Biochemistry 47:421-30
La Mar, Gerd N (2007) Application of the paramagnetic dipole field for solution NMR active site structure determination in low-spin, cyanide-inhibited ferric hemoproteins. IUBMB Life 59:513-27

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