Enhancing Expression of Recombinant Hemoproteins: Progress Toward Understanding Structure/Function and Therapeutic Application
Type of DegreeDissertation
Chemistry and Biochemistry
MetadataShow full item record
Nature has developed ways to control the essential need to activate oxygen. Hemoproteins perform this task using iron in a prosthetic group (e.g., iron-protoporphyrin-IX or heme). The environment surrounding the heme iron in hemoproteins dictates their activity and makes them a diverse group of enzymes. This makes hemoproteins good models for exploring the relationship between protein structure and function. The techniques necessary to pursue these studies are material-intensive. Commonly available systems for the expression of recombinant hemoproteins in E. coli are able to produce large quantities of protein. However, in many cases the vast majority of the protein lacks heme and are therefore inactive. A hemoprotein expression (HPEX) system was developed to resolve this problem. This system, based on the expression of an outer membrane heme receptor, was tested on two different hemoproteins, myoglobin and catalase-peroxidase. In both cases, the system was successful, demonstrating a dramatic increase in the heme content of these proteins. Additional studies were carried out on the catalase-peroxidases. The ability of catalase-peroxidases to catalyze catalase and peroxidase activities using one active site make them ideal for studying the protein structure/function relationship. Moreover, a periplasm-targeted subset of these enzymes have been implicated as virulence factors. The first full characterization of a periplasmic catalase-peroxidase was carried out on KatP, an enzyme from the highly virulent pathogen E. coli O157:H7. Absorption and EPR spectra indicated a high-spin heme enzyme dominated by the hexacoordinate high-spin complex. Apparent kcat values for catalase and peroxidase activities were higher for KatP than with other catalase-peroxidase. However, KM values were also higher for KatP. Ferric KatP reacted with peracetic acid to form compound I and with CN- to form a ferri-cyano complex consistent with other catalase–peroxidases. Peroxynitrite also supported compound I formation in this enzyme. This catalytic capability, combined with efficient catalase and peroxidase activities, and a periplasmic location may be advantageous for meeting an immune response that generates copious reactive oxygen and reactive nitrogen species. Crystal structures of catalase-peroxidases have revealed the presence of a three amino acid covalent adduct (Trp-Tyr-Met). It is essential for catalase activity. An SDS-PAGE method to monitor the establishment of the crosslink was developed. Using this method it was demonstrated that crosslink formation requires a redox active porphyrin. Substitution of the Tyr residue with Phe prevent formation of the crosslink and generates a protein that has no catalase activity, enhanced peroxidase activity, and increased susceptibility to peroxide-dependent inactivation.