The work described in this proposal is an extension of our continuing effort to describe and compare the active site dynamics of two unique heme proteins. One of these is cytochrome c peroxidase, a ferriheme enzyme isolated from baker's yeast in the laboratory of Dr. James Erman. The other protein is actually a group of proteins which have been individually isolated and purified in my own laboratory and which comprise the so called """"""""Monomer Hemoglobin"""""""" fraction of Glycera Dibranchiata. Cytochrome c peroxidase (CcP) has been studied by less precise spectroscopic methods, but our initial work has shown that proton nuclear magnetic resonance spectroscopy can be used effectively to identify the role played by specific groups, both on the protein and on the heme group, which influence the enzyme's reactivity (vide infra, see also the reprints and preprints). We plan to continue our dynamics studies with nmr employing saturation transfer and Redfield pulse sequence experiments. These will result in further assignments of hyperfine shifted protons, analysis of heme binding dynamics, elucidation of the lability of hyperfine shifted protons and quantitation of the extent of communications between extrinsic molecules and the individual atoms which are components of the active site. Our work on the second group of proteins is on the verge of rapid expansion during the present year due to our efforts during the recent past at purifying the Glycera monomer fraction. Of prime importance in this effort is the fact that one of these components has been determined to lack the distal histidine. Studies of these proteins will include ligation dynamics, equilibrium ligation studies and these will be related to the spectroscopic characterization of the heme pocket in conjunction with sequence and structural studies carried out elsewhere. Conclusions drawn from these studies will have extensive impact upon theories of the particular role which distal residues play in both cooperative and non-cooperative ligand binding processes. These will be true structure-function correlations which may define specific factors which govern the primary process (ligand binding) of non-covalent heme proteins. The results of this research will impact the design of synthetic heme enzymes and tailored heme proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK030912-08
Application #
3229734
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1989-09-01
Project End
1991-08-31
Budget Start
1989-09-01
Budget End
1991-08-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Washington State University
Department
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Aiken, N R; Galey, W R; Satterlee, J D (1995) A peroxidative model of human erythrocyte intracellular Ca2+ changes with in vivo cell aging: measurement by 19F-NMR spectroscopy. Biochim Biophys Acta 1270:52-7
Aiken, N R; Satterlee, J D; Galey, W R (1992) Measurement of intracellular Ca2+ in young and old human erythrocytes using 19F-NMR spectroscopy. Biochim Biophys Acta 1136:155-60
Satterlee, J D; Russell, D J; Erman, J E (1991) Proton homonuclear correlated spectroscopy as an assignment tool for hyperfine-shifted resonances in medium-sized paramagnetic proteins: cyanide-ligated yeast cytochrome c peroxidase as an example. Biochemistry 30:9072-7
Satterlee, J D; Erman, J E (1991) Proton NMR assignments of heme contacts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects. Biochemistry 30:4398-405
Satterlee, J D; Erman, J E; Mauro, J M et al. (1990) Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant. Biochemistry 29:8797-804
Alden, R G; Satterlee, J D; Mintorovitch, J et al. (1989) The effects of high pressure upon ligated and deoxyhemoglobins and myoglobin. An optical spectroscopic study. J Biol Chem 264:1933-40
Constantinidis, I; Kandler, R L; Satterlee, J D (1989) Purity of Glycera dibranchiata monomer hemoglobin components III and IV determined by isoelectric focusing. Comp Biochem Physiol B 92:619-22
Mintorovitch, J; van Pelt, D; Satterlee, J D (1989) Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV. Biochemistry 28:6099-104
Constantinidis, I; Satterlee, J D; Pandey, R K et al. (1988) Assignment of selected hyperfine proton NMR resonances in the met forms of Glycera dibranchiata monomer hemoglobins and comparisons with sperm whale metmyoglobin. Biochemistry 27:3069-76
Mintorovitch, J; Satterlee, J D (1988) Anomalously slow cyanide binding to Glycera dibranchiata monomer methemoglobin component II: implication for the equilibrium constant. Biochemistry 27:8045-50

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