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Roles of an 'Inactive' Domain in Catalase-Peroxidase Catalysis: Modulation of Active Site Architecture and Function by Gene Duplication




Hartfield-Baker, Ruletna

Type of Degree



Chemistry and Biochemistry


Within biological systems iron is a transition metal that allows access to the benefits of molecular oxygen as an oxidant. However, with these benefits come grave consequences if the reactions are not strictly controlled. The most prominent strategy of control and specialization is the protein environment that surrounds iron. Within iron containing proteins, specifically heme proteins, there are four basic levels of structure that impact the iron’s function: cofactor structure, protein-supplied ligands, non-ligand active site environment, and protein features that are distant from the active site. This last level remains poorly understood due to a lack of good models to pursue such studies. Catalase-peroxidases are unique heme proteins because they catalyze peroxide decomposition by two separate mechanisms, catalase and peroxidase, using the same active site. However, were it not for three structural features distant from the active site, catalase-peroxidases would be practically superimposable with monofunctional peroxidases. Given the additional reactivity of catalase-peroxidases it seems probable that these features would be involved in the function and would, therefore, provide excellent models for understanding the roles of features distant from the active site. The largest of these three features, the C-terminal domain, about 300 amino acids, is situated ? 30 Å from the active site. In order to determine its role, we produced a variant of E. coli catalase-peroxidase (KatG) lacking its C-terminal domain (KatGNterm). KatGNterm was expressed in inclusion bodies and required us to develop a denatured purification procedure, as well as refolding and reconstitution protocols in order to isolate it. A soluble, well-behaved KatGNterm, was produced; however, it had neither catalase nor peroxidase activity. Far-UV CD and UV-visible spectroscopy confirmed that the protein was correctly folded, and that heme was bound within the active site. Visible absorption and EPR spectra indicated a shift from the predominately high-spin pentacoordinate heme environment to an exclusively hexacoordinate low-spin environment. These resultss were also observed in the EPR spectrum of KatGNterm. Peroxyacetic acid and cyanide binding experiments revealed what seem to be an occluded active site. Site-directed substitution of KatGNterm active site histidine residues with alanine showed that His106, normally required as a general base, had become a ligand to the heme. Reincorporation of a separately expressed an isolated C-terminal domain, KatGCterm, resulted in recovery catalase and peroxidase activity. Both EPR and UV-visible spectroscopy confirmed the restoration of the overall architecture of the active site. This research demonstrates that the C-terminal domain is essential for activity by maintaining the correct coordination environment for the heme, even though it is some 30 Å away from the active site.