Of the known biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. Rapid progress in recent years as revealed a P450 gene superfamily with numerous members in bacteria, fungi, plants, invertebrates, and vertebrates, including the human. These enzymes are of great interest from a fundamental point of view because of their remarkable versatility and reaction mechanisms involving the generation of a powerful oxidant from molecular oxygen, and also from the viewpoint of their biomedical relevance. Because the substrates include physiologically important compounds such as steroids, bile acids, fatty acids, prostaglandins, retinoids, biogenic amines, and lipid hydroperoxides, as well as a host of """"""""environmental chemicals,"""""""" it is no exaggeration to say that improved knowledge of P450 function will contribute to progress in drug metabolism and design, as well as to better insights into chemical carcinogenesis, alcoholism, endocrine disorders, and oxidative stress. Our main objectives are: 1. To obtain detailed evidence on the important question of whether multiple oxygenating species contribute to substrate oxidation by cytochrome P450. Mutant P450s blocked in proton delivery to the active site will be examined for their rates of oxidation of various substrates as an indication of the role of peroxo-hydroperoxo- and oxenoid-iron as discrete oxidants. Attempts will be made to characterize these labile species by chemical and physical methods in order to correlate the apparent steady state level of a particular oxidant with the catalytic rates. In addition, the possibility will be examined that the levels of the oxidants are subject to regulation by effectors, including cytochrome b5 and flavonoids. 2. To continue our attempts to obtain one or more mammalian P450s in a crystalline form suitable for structure determination by x-ray diffraction. Various full-length and truncated P450s will be studied for this purpose. 3. To determine the role of phospholipids and other membrane components in influencing the formation of binary and ternary complexes of P450, NADPH-P450 reductase, and cytochrome b5, and in altering the rates and specificities of substrate oxidation in lipid bilayers. The effect of lipid composition in membrane bilayers. The effect of lipid composition in membrane bilayers will be examined with respect to single-phase fluid membranes with co-existing solid-phase obstacles to protein diffusion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK010339-34
Application #
2859194
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Sechi, Salvatore
Project Start
1976-04-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
34
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Newcomb, Martin; Lansakara-P, Dharmika S P; Kim, Hye-Yeong et al. (2007) Products from enzyme-catalyzed oxidations of norcarenes. J Org Chem 72:1128-33
Newcomb, Martin; Chandrasena, R Esala P; Lansakara-P, Dharmika S P et al. (2007) Desaturase reactions complicate the use of norcarane as a mechanistic probe. Unraveling the mixture of twenty-plus products formed in enzyme-catalyzed oxidations of norcarane. J Org Chem 72:1121-7
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; 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
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

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