The overall goal of the research project is an improved understanding of the structure-function relationships and control mechanisms for mammalian cytochrome P-450. Such information is essential for an adequate evaluation of the role of this large and versatile family of cytochromes in the metabolism of steroids and prostaglandins and in the conversion of environmental chemicals either to more toxic substances, including carcinogens, or to detoxified products.
The specific aims are as follows: (a) to isolate and characterize the remaining unidentified or inadequately identified P-450 isozymes of rabbit liver and determine their physical and chemical properties, including amino acid sequences. The cytochromes to be purified include one for which this laboratory has already obtained the predicted sequence from an isolated gene (P-450z) and other P-450's that are induced by clofibrate, flavonoids, cholestyramine, debrisoquine, or starvation, are sex-specific, or are responsible for the oxygenation of steroids and prostaglandins in various positions. Attempts to crystallize these membrane-bound enzymes for the purpose of X-ray diffraction will be continued, and additional genes, particularly of the """"""""phenobarbital family,"""""""" will be isolated and characterized. A related goal is to carry out site-specific mutagenesis of selected P-450 amino acid residues that are likely to play a role in P-450 catalysis or the binding of substrates and electron donors; (b) to determine the substrate specificity of liver microsomal P-450's under conditions more typical of the in vivo situation, with partially limiting substrate and electron carrier concentrations. The rates of competing reactions in which 0-2 is converted to H-20-2 or undergoes 4-electron reduction with the presumed formation of water will also be determined; and (c) to study factors that control the activity of the P-450 system, such as induction and repression, possible allosteric effectors, and cytochrome b-5. The effects of b-5 are striking in that it stimulates some reactions and inhibits others. The mechanism, which apparently involves electron transfer to or from b-5 by the Fe-II-0-2 complexes of the P-450 isozymes, will be studied by stopped flow spectrophotometry. Preliminary results indicate that b-5 reacts differently with different P-450's and controls the rate of formation of several other oxygen-containing species from the ferrous dioxygen complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK010339-22
Application #
3482942
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1976-04-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
22
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Vatsis, Kostas P; Coon, Minor J (2005) Oxidative aldehyde deformylation catalyzed by NADPH-cytochrome P450 reductase and the flavoprotein domain of neuronal nitric oxide synthase. Biochem Biophys Res Commun 337:1107-11
Vatsis, Kostas P; Peng, Hwei-Ming; Coon, Minor J (2005) Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4. Arch Biochem Biophys 434:128-38
Chandrasena, R Esala P; Vatsis, Kostas P; Coon, Minor J et al. (2004) Hydroxylation by the hydroperoxy-iron species in cytochrome P450 enzymes. J Am Chem Soc 126:115-26
Newcomb, Martin; Aebisher, David; Shen, Runnan et al. (2003) Kinetic isotope effects implicate two electrophilic oxidants in cytochrome p450-catalyzed hydroxylations. J Am Chem Soc 125:6064-5
Newcomb, Martin; Hollenberg, Paul F; Coon, Minor J (2003) Multiple mechanisms and multiple oxidants in P450-catalyzed hydroxylations. Arch Biochem Biophys 409:72-9
Vatsis, Kostas P; Peng, Hwei-Ming; Coon, Minor J (2002) Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity. J Inorg Biochem 91:542-53
Newcomb, Martin; Shen, Runnan; Lu, Yun et al. (2002) Evaluation of norcarane as a probe for radicals in cytochome p450- and soluble methane monooxygenase-catalyzed hydroxylation reactions. J Am Chem Soc 124:6879-86
Vatsis, Kostas P; Coon, Minor J (2002) Ipso-substitution by cytochrome P450 with conversion of p-hydroxybenzene derivatives to hydroquinone: evidence for hydroperoxo-iron as the active oxygen species. Arch Biochem Biophys 397:119-29
Raner, G M; Hatchell, A J; Morton, P E et al. (2000) Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes. J Inorg Biochem 81:153-60
LeLean, J E; Moon, N; Dunham, W R et al. (2000) EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding. Biochem Biophys Res Commun 276:762-6

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