Oxygen biochemistry associated with hemeproteins will be investigated. Experiments designed to identify and characterize reactions that generate hydrogen peroxide and/or oxyradicals, reactions that result in heme oxidation and protein modification by hydrogen peroxide and/or oxyradicals, and reactions of the oxidase, proton pumping, and carbon monoxide dioxygenase functions of cytochrome c oxidase (CcO) will be carried out. Rate and product studies of reactions of oxyhemeproteins with oxidant drugs and structurally related reductants to give oxidized heme iron, H2O2, and one-electron oxidized reductant (usually a free radical) will consider the dependence of rate upon the structures of both protein and reductant and the most likely mechanisms for these reactions. Studies of heme oxidation during autoxidations will focus on participation of H2O2 and oxyradicals, the dependence of reaction rates on protein structure, and effects of superoxide dismutase, catalase, glutathione, ascorbate and Alpha-tocopherol. Modifications to amino acid residues of hemeproteins that occur in solutions when H2O2 or oxyradicals are present will be explored to ascertain the specific reactions involved. Normal and abnormal human hemoglobins (e.g., A,F,S,C and Zurich) and myoglobins from human and bovine hearts are the proteins to be used most widely in the above studies with some exploratory experiments utilizing bovine heart CcO. The possibility that certain hemoglobins (e.g. S,C,Zurich) from different individuals may consist of subpopulations of protein molecules which are modified differently on exposure to oxidant stress will be tested. All or many of the reactions considered may occur simultaneously in a given hemeprotein solution and are important in normal aging as well as during conditions of oxidant stress. The principles of O2 biochemistry discovered will also apply to O2-utilizing enzymes. The structure and reactions of bovine heart CcO will be studied to define roles for metals (Fe, Cu, Zn, Mg) and to elucidate structural features that are retained among oxidases from different species and appear essential to the O2-reduction and H+-pumping function of the oxidase. Spectroscopic methods (uv-visible, IR, NMR) will be used to monitor reaction rates and to characterize reactants and products. HPLC and other chromatographic methods will be used extensively in the isolation, separation, and identification of proteins, peptides, and amino acids.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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Colorado State University-Fort Collins
Schools of Arts and Sciences
Fort Collins
United States
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Dong, A; Caughey, W S (1994) Infrared methods for study of hemoglobin reactions and structures. Methods Enzymol 232:139-75
Dong, A; Nagai, M; Yoneyama, Y et al. (1994) Determination of the amounts and oxidation states of hemoglobins M Boston and M Saskatoon in single erythrocytes by infrared microspectroscopy. J Biol Chem 269:25365-8
Dong, A C; Huang, P; Caughey, W S (1992) Redox-dependent changes in beta-extended chain and turn structures of cytochrome c in water solution determined by second derivative amide I infrared spectra. Biochemistry 31:182-9
Yoshikawa, S; Caughey, W S (1992) Infrared evidence of azide binding to iron, copper, and non-metal sites in heart cytochrome c oxidase. J Biol Chem 267:9757-66
Caughey, B W; Dong, A; Bhat, K S et al. (1991) Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry 30:7672-80
Lee, H C; Booth, K S; Caughey, W S et al. (1991) Interaction of halides with the cyanide complex of myeloperoxidase: a model for substrate binding to compound I. Biochim Biophys Acta 1076:317-20
Young, L J; Caughey, W S (1990) Pathobiochemistry of CO poisoning. FEBS Lett 272:1-6
Yoshikawa, S; Caughey, W S (1990) Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction. J Biol Chem 265:7945-58
Dugad, L B; La Mar, G N; Lee, H C et al. (1990) A nuclear Overhauser effect study of the active site of myeloperoxidase. Structural similarity of the prosthetic group to that on lactoperoxidase. J Biol Chem 265:7173-9
Dong, A; Huang, P; Caughey, W S (1990) Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry 29:3303-8

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