Delineation of the active site structure of chloroperoxidase from C. fumago

Chloroperoxidase, a heme-containing glycoprotein from Caldariomyces fumago, is secreted in prodigious amounts in the presence of fructose. Production of 90% pure, mycelium-free enzyme for 200 days from a single inoculum was facilitated by continuous and semi-continuous bioreactors designed and built during the course of this thesis. Chloroperoxidase defies common notions of enzyme specificity by catalyzing a broad spectrum of reactions characteristic of other distinct classes of heme proteins. Previous investigations have focused primarily on the characterization of the various reactivities and oxidized intermediates of the enzyme. As detailed in this thesis, studies were carried out to identify active site features that are important in facilitating catalysis. The proximal ligand is important towards dictating the catalytic, spectroscopic, and redox properties of hemeproteins. Strong evidence for thiolate heme ligation in chloroperoxidase had been previously derived based on spectroscopic data. Chloroperoxidase possesses 3 cysteine residues, two of which are involved in a disulfide linkage. The apoenzyme was incubated with the sulfhydryl specific reagent DTNB, and subsequently digested with endoproteinase-glu, resulting in a single modified cysteinyl peptide isolated via reverse phase HPLC. Edman sequence analysis explicitly identified the heme axial fifth ligand of chloroperoxidase as cysteine 29. Electroreduction of the protein's disulfide linkage via controlled potential electrolysis yielded evidence that the integrity of the covalent bond is necessary for enzyme activity. Resonance Raman studies indicated that chloroperoxidase is predominantly a high spin, pentacoordinate complex between the pH values of 2 to 7. The structure of the distal side of the chloroperoxidase heme pocket was also examined. Previous investigations had led to the proposal by a number of investigators that chloroperoxidase possessed a distal histidine, similar to the invariant residue found in the active site of both peroxidases and catalases. To address this postulate in a more direct manner, chemical modification studies were commenced with the reagent diethylpyrocarbonate. It was found that when chloroperoxidase was incubated with excess diethylpyrocarbonate at near neutral pH values, inactivation of enzyme occurred due to the modification of a single essential histidine residue. Sequence homology with cytochrome c peroxidase led to the proposal that residue 38 may be a candidate for the putative distal histidine of chloroperoxidase. Secondary structure predictions allowed for the design of an active site peptide that included cysteine 29 and histidine 38.U of I OnlyETDs are only available to UIUC Users without author permissio