Biochemistry and Hepatic Toxicity of Carbon Radicals

Investigator: Paul R. Ortiz De Montellano, PhD
Sponsor: NIH National Institute of General Medical Sciences

Location(s): United States


Free radical processes have been implicated in a growing range of pathologies, including major health problems such as cancer and atherosclerosis. Nevertheless, despite impressive progress in the past few years, the basic biochemistry of protein radicals continues to be incompletely understood. The work proposed rests on key advances made in the expiring period of support, including (a) identification of the residues in autocatalytic covalent binding of the heme group in lactoperoxidase, (b) Identification of specific residues in lactoperoxidase that are converted to free radicals centers upon reaction of the enzyme with H202, (c) mutation of horseradish peroxidase into an enzyme that mimics the mammalian peroxidases in its ability to bind its heme covalently, and (d) demonstration in a metmyoglobin system that the oxidation of tyrosines to radicals depends on factors other than distance from the oxidizing center. The present project focuses on two aspects of protein radical biochemistry: (a) the possible formation and role of carboxylate radicals, a highly neglected species, in peroxidase function, and (b) the mechanism of formation of unusual intramolecular cross-links and their functional consequences. The first aim of the present project is to define the role of the carboxylate group in the heme-protein cross-linking reaction through studies of the horseradish peroxidase model system. The second aim is to use the same model to clarify the role of radicals in formation of the methionine-vinyl bond of myeloperoxidase. The third aim is to clarify why covalent heme binding is important for mammalian peroxidase function. The fourth aim is to examine the role of the active site carboxylate group and the mechanism(s) by which the unusual cross-links in catalase-peroxidase enzymes are formed. A final goal is to employ a metmyoglobin model system with appended phenols to explore the relationships which determine which tyrosines are oxidized in a protein. These studies should shed light on the generation and fates of peroxidatively generated carbon radicals in physiological and pathological processes and provide insights into approaches for the modulation or suppression of such processes.