We propose the detailed study of functionally relevant molecular/electronic structural and dynamic properties of a series of heme oxygenase, HO, enzymes and their complexes with substrate/reaction intermediates in variable oxidation/spin/liagtion states, using high resolution solution 2D/3D NMR. HO, found in vertebrates, plants and bacteria, acts by a common mechanism and set of intermediates, using heme as both substrate and cofactor, to stereoselectively cleave heme into a-biliverdin, iron and CO. HO is unique in using the hydroperoxy species as its activated form, and the structural properties of the active site that stabilize the species are not well understood except that ordered water molecules within a distal H-bond network are involved. We select 3 HOs, isozyme #1 from human, hHO, and those from 2 pathogenic bacteria C. diphtheriae (CofHO) and N. meningitidis (NmHO), which share a common fold, but exhibit variable sequence homology for the residues involved in the H-bonding network. The target derivatives are substrate-free or app-HO, resting state HO-hemin-H/jO, HO-hemin-CN as a model for the unstable oxy complex, and HO-hemin-OH as a model for the reactive hydroperpxy species. Since all but 1 targeted HO derivative are paramagnetic, emphasis is placed on utilizing appropriately tailored 1D/2D/3D NMR to extract the wealth of unique information in hyperfine shifts. We will develop a new and highly sensitive NMR probe that directly reflect the degree of H-bonding between axial ligand to the hemin and the distal ordered-water/H-bond network, using the pair of complex HO-hemin-H2O/-OH, and to use this probe, as well as previously established procedures, to provide a detailed characterization of the solution structure. Our interests focus on comparison of local solution with cryogenic crystallographic molecular structure, with particular attention paid to extended H-bond networks with some remarkably robust H-bonds, and the ordered water molecules within these networks. We emphasize comparative studies among the various derivatives of 1 HO, and among the different HOs for a given derivative, to elucidate the relationship between variable strength H-bonds and axial ligand properties. In addition, we will characterize the influence of HO, substrate or intermediates and their axial ligands on dynamic properties related to entry and exit of substrate. Lastly, for NmHO, we will characterize the influence of heme substituents on its seating in the active site, determine the structure of the crystallographically disordered C-terminus found folded into the active site in solution, and illuminate the role of the C-terminus and the unique active site Cys113 in multiple, functionally relevant, microheterogeneities. The detailed description of the molecular structural and dynamic properties of heme oxygenase will improve our understanding of the varied roles of mammalian enzymes. The elucidation of the similarities and differences between bacterial and mammalian heme oxygenase will improve prospects for the design of selective inhibitors for the enzyme in pathogenic bacteria. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062830-06
Application #
7230507
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Wehrle, Janna P
Project Start
2001-05-01
Project End
2010-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
6
Fiscal Year
2007
Total Cost
$214,687
Indirect Cost
Name
University of California Davis
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
047120084
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
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; 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
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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