Structur-function relationships of human peroxidases

The homologous mammalian peroxidase superfamily II includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO). MPO and EPO (located in granules of neutrophils and eosinophils, respectively) and LPO (found in secretion fluids such as milk and saliva) play a central role in the antimicrobial defense system, whereas TPO is involved in the biosynthesis of thyroid hormones. MPO and EPO have also been implicated in promoting tissue damage in numerous inflammatory diseases. Despite the physiological relevance and molecular and enzymatic peculiarities of human peroxidases, a comprehensive study of the specific role of individual amino acids in the formation and reactivity of redox intermediates involved in catalysis is still missing. This will be done in this project, which combines the genetic experience of Nicole Moguilevsky`s group (who has been successful in the production of unprocessed recombinant MPO and mutants from Chinese hamster ovary cell lines) and of our group (who has been successful in the massive purification of mature MPO and EPO from human blood as well as in establishing protocols for transient kinetic investigations of these enzymes). Based on our recent common paper (Furtmüller et al., FEBS Lett. (2001) 503, 147-150) that the recombinant unprocessed monomeric MPO shows essentially the same kinetic behaviour as the mature dimeric protein, as well as on the known structure of MPO and theoretical models for both EPO and LPO, the production of about 40 mutants of recombinant MPO is suggested. The effect of mutation on ligand binding, redox properties and formation, stability and reactivity of compounds I and II will be investigated in order to answer the following questions: What is the effect of the extraordinary nature of the heme (and its covalent and non-covalent interactions with the protein) on the reactivity of the individual enzyme intermediates and in consequence on the different substrate specificity of MPO, EPO and LPO? What controls the redox properties of the ferric protein and of compounds I and II? What is the actual halide binding site? What controls the potential interaction and reactivity towards organic one-electron donors? What is the proton balance in compound I and II reduction and why is compound II reduction (in contrast to compound I reduction) of human peroxidases strongly constrained by the nature of the substrate? What is the physiological role and the actual site of protein radical formation observed with human peroxidases?

Members of the human heme peroxidase family include eosinophil peroxidase (EPO), lactoperoxidase (LPO), and myeloperoxidase (MPO), which produce a complex array of reactive oxidants that mediate halogenation, nitro(syl)ation and oxidation of cellular components such as lipids and proteins. Therefore, these peroxidases have - beside their important role in the innate immune defence system - enormous potential to inflict tissue damage and to induce inflammation. Thus they are the up-and-coming area in inflammation research, and will lead to the development of drugs targeted to reduce pathology in diseases such as cardiovascular disease, neurodegenerative disorders and asthma. The ability to limit inflammation by targeting peroxidases depends on building a thorough understanding of the structure and function of these enzymes and this was the aim of this project. In the present project for the first time the spectral, kinetic and redox properties of all (short-living) enzyme intermediates (i.e. ferric and ferrous proteins, compounds I, II & III) of MPO and several mutants, EPO and LPO have been elucidated in a comparative study. A new method for the determination of reduction potentials of all short-living redox intermediates involved in the halogenation and peroxidase cycle of these heme peroxidases has been developed. Furthermore, the sequence and kinetics of reactions of interconversion between ferrous MPO/LPO and compound III as well as between ferrous MPO/LPO and compound II and compound III have been elucidated allowing to clarify the catalatic activity of mammalian peroxidases, which was long under discussion. By using side-directed mutagenesis the role of the heme to protein linkages in catalysis have been investigated. The unusual covalent links are responsible for the asymmetric bow-shaped structure of the heme and the peculiar spectroscopic properties. Disruption of the protein-heme linkages increased the flexibility of the prosthetic group thereby lowering the reduction potential of compound I and decreasing the capicity in chloride and bromide oxidation. The peculiar role of the MPO-typical sulfonium linkage in chloride oxidation by compound I could be demonstrated. Neighboured Glu242 was shown to be important in halide binding and its exchange led to a decrease in the oxidation rates of all (pseudo-) halides whereas a cleaved Asp94 link exhibited a slightly reduced chloride and bromide but not iodide and thiocyanate oxidation rate and had no impact on MPO oxidation by hydrogen peroxide (i.e. compound I formation).