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|Title:||Characterization of the Active Site of Chloroperoxidase Using Physical Techniques (Hemeproteins)|
|Author(s):||Hall, Kathleen Southwick|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of my research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins.
To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary ('1)H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D(,2)O media with a small amount of ('1)H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the ('1)H-NMR spectra of CO-CPO (proto enzyme) and CO-CPO (labeled enzyme) indicate the presence of a cysteine ligand in chloroperoxidase.
Sulfur was replaced by selenium in CPO for ('77)Se-NMR and ENDOR experiments. In media containing MgSO(,4) and MgSeO(,4), C. fumago incorporates selenium into CPO methionine residues but not into cysteine. Resonance Raman of this selenium rich sample shows no change in the Fe-S frequency mode indicating that methionine is not a ligand to the iron.
EXAFS experiments on native CPO (Fe('+3)) and on a very unstable intermediate Compound (I) have been completed. For the Compound (I) experiment a technique was developed which enabled us to do EXAFS on freeze-quenched samples. This data confirms previous Mossbauer results. Future resonance Raman and EXAFS experiments may be able to determine the bond length of the Fe-O bond of Compound (I). Native CPO EXAFS results of high spin, low spin and intermediate states enabled Dr. M. Chance of Bell Laboratories to improve on mixed spin state calculations which relate more directly to mechanistic studies of hemeproteins. (Abstract shortened with permission of author.)
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1986.
|Date Available in IDEALS:||2014-12-15|