The research will apply methods from synthetic and physical inorganic chemistry, as well as more traditional biochemical methods, to selected proteins and enzymes containing transition metals and flavins. The general objective is to understand the relationship between the structure of metal- and flavin-containing systems and their biological function. This will be accomplished by fundamental physical, chemical and biochemical studies of representative members of a novel class of metal-containing hydrolytic enzyme, and by the preparation and study of synthetic systems containing flavins and iron-sulfur clusters as models for complex metalloflavoenzymes. The specific examples to be investigated are members of the novel and biologically widespread class of purple metallophosphatases, an unusual green heme peroxidase from bovine spleen, and models for metal-flavin enzymes. The purple acid phosphatase from bovine spleen has been shown to contain a mixed-valence binuclear iron cluster at the active site, while a similar enzyme from sweet potato is reported to contain manganese. These enzymes, along with a representative purple phosphatase from a microbial source, will be examined by spectroscopic and chemical methods to determine the nature of the metal prosthetic groups and their role in catalyzing a simple hydrolysis reaction. Biochemical and immunological techniques will be used to investigate the physiological function of the bovine spleen enzyme in vivo; preliminary indications are that it is a phosphoprotein phosphatase specific for phosphotyrosine and that it may be involved in regulation of other enzymes in the immune system. The structure and function of the unusual chromophore in the green heme peroxidase from bovine spleen will be examined by resonance Raman and NMR spectroscopy. Electron transfer reactions between iron-sulfur clusters and flavin will be examined, using systems containing flavins covalently attached to iron-sulfur clusters via spacers of different length to determine intramolecular electron transfer rates and the extent of magnetic interactions. Cytochrome c covalently modified with flavin derivatives will be prepared and used to measure flavin-heme electron transfer rates.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032117-09
Application #
3280733
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1983-01-01
Project End
1993-04-30
Budget Start
1991-05-01
Budget End
1993-04-30
Support Year
9
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Orlando, J L; Zirino, T; Quirk, B J et al. (1993) Purification and properties of the native form of the purple acid phosphatase from bovine spleen. Biochemistry 32:8120-9
Averill, B A; Vincent, J B (1993) Electronic absorption spectroscopy of nonheme iron proteins. Methods Enzymol 226:33-51
Wang, Z; Ming, L J; Que Jr, L et al. (1992) 1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen. Biochemistry 31:5263-8
Vincent, J B; Crowder, M W; Averill, B A (1992) Multiple binding sites for tetrahedral oxyanion inhibitors of bovine spleen purple acid phosphatase. Biochemistry 31:3033-7
Vincent, J B; Crowder, M W; Averill, B A (1992) Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions. Trends Biochem Sci 17:105-10
Crowder, M W; Vincent, J B; Averill, B A (1992) Electron paramagnetic resonance studies on the high-salt form of bovine spleen purple acid phosphatase. Biochemistry 31:9603-8
Vincent, J B; Crowder, M W; Averill, B A (1991) Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen. J Biol Chem 266:17737-40
Vincent, J B; Crowder, M W; Averill, B A (1991) Spectroscopic and kinetics studies of a high-salt-stabilized form of the purple acid phosphatase from bovine spleen. Biochemistry 30:3025-34
Vincent, J B; Averill, B A (1990) An enzyme with a double identity: purple acid phosphatase and tartrate-resistant acid phosphatase. FASEB J 4:3009-14
Vincent, J B; Averill, B A (1990) Sequence homology between purple acid phosphatases and phosphoprotein phosphatases. Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers? FEBS Lett 263:265-8

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