Towards a Unified View of Substrate Binding in Heme Peroxidases.

Heme peroxidases catalyse the H2O2-dependent oxidation of a variety of substrates. The binding of substrates to heme peroxidases has been widely assumed to occur at the so-called δ-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the γ-heme edge, is also possible. In this thesis substrate binding in two class I peroxidases, cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), has been examined using a variety of methods with the aim of providing a more unified view of substrate binding across the family of heme peroxidases. It has been shown that the closely related CcP enzyme can duplicate the substrate binding properties of APX through the introduction of relatively modest structural changes. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of CcP is presented showing ascorbate bound to the γ-heme edge. Functional studies have shown that a transient porphyrin π-cation radical in CcP, analogous to that observed in APX, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The first crystal structures of complexes of the tuberculosis prodrug, isoniazid, bound to APX and CcP are presented along with the structures of the isoniazid complexes of two active site mutants of APX. These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases and provide rationalization of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis. Aromatic substrate binding in CcP has been examined by X-ray crystallography and competitive inhibition studies and directed evolution has been used to probe aromatic substrate binding in APX. Finally, the first neutron diffraction structure of CcP has been solved allowing a detailed description of the resting ferric enzyme and showing that it would be a viable technique for use of studying the nature of key intermediates and gaining further insight into enzymesubstrate interactions. Understanding the structural and chemical requirements for efficient substrate binding and oxidation by heme peroxidases will provide a more in-depth understanding of the mechanism they use and will pave the way for the production of enzymes with improved catalytic efficiency and novel functions and the development of improved therapeutic agents against diseases such as tuberculosis.