Mechanisms of molecular oxygen and peroxide activation by the nitric oxide synthases and the heme degrading enzyme ChuS

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The long term goal of my research program is to detail at the molecular level the kinetic mechanisms of heme enzymes, i.e. enzymes that use heme as a cofactor. Heme enzymes play numerous important functions such as the degradation of xenobiotics, synthesis of sexual hormones, bioremediation, lignin degradation, etc... We propose to decipher the reaction mechanism of two heme enzymes involved in extremely important processes in bacteria (ChuS and NOS) and humans (NOS). ChuS is an enzyme that degrades heme. It part of the heme internalisation and utilisation pathway that is largely distributed in environmental and pathogenic bacteria (Shigella, E. coli, and others) that allows these bacteria to use heme as a source of iron for growth. ChuS and ChuS-homologues are absent in human and animals. This enzyme may thus become additional targets in the fight against pathogenic bacterial infections. NOSs are heme enzymes that synthesize the gaseous radical nitric oxide (NO) that has dual functions as a messenger and cytotoxic molecule produced in human. NO is involved in many aspects of human physiology (control of blood pressure, immune response, neural communication) and diseases including atherosclerosis, neurodegenerative diseases, diabetes and cancer. NOSs are also found in a subset of bacteria where they exhibit surprisingly diverse but critical functions. Both ChuS and NOS are heme enzymes that must activate molecular oxygen and/or hydrogen peroxide to carry out their function, which involves oxidation reactions. We propose to decipher the detailed kinetic mechanism by which NOS and ChuS carry out the oxidation of their substrates through the use of various biophysical techniques that will allow the determination of reaction rates of each kinetic step of the enzymatic process and the identification of the nature of the kinetic intermediates. These studies are critical to understand the role of ChuS and NOS in physiology and disease and for the design of potent and specific inhibitors. These studies should be of general interest to those interested in redox reactions carried out by enzymes in academia, industry, pharmaceutical and biotechnology sectors.