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

我的研究计划的长期目标是在分子水平上详细说明血红素酶的动力学机制，即使用血红素作为辅因子的酶。血红素酶在生物降解外源性物质、合成性激素、生物修复、降解木质素等方面发挥着重要作用。我们建议破译两个血红素酶参与细菌（ChuS和NOS）和人类（NOS）中极其重要的过程的反应机制。ChuS是一种降解血红素的酶。它是血红素内化和利用途径的一部分，该途径主要分布在环境和病原细菌中（志贺氏菌，大肠杆菌，志贺菌，志贺菌）。大肠杆菌和其他细菌），使这些细菌能够利用血红素作为生长的铁源。ChuS和ChuS同源物在人类和动物中不存在。因此，这种酶可能成为对抗病原性细菌感染的额外靶标。一氧化氮合酶（NOS）是一种血红素酶，能合成一氧化氮（NO），具有信使和细胞毒双重功能。NO参与人体生理学的许多方面（血压控制、免疫反应、神经通讯）和疾病，包括动脉粥样硬化、神经退行性疾病、糖尿病和癌症。NOS也存在于一个细菌亚群中，它们表现出惊人的多样性，但关键的功能。ChuS和NOS都是血红素酶，它们必须激活分子氧和/或过氧化氢来执行它们的功能，这涉及氧化反应。我们建议破译详细的动力学机制，NOS和ChuS进行其底物的氧化，通过使用各种生物物理技术，将允许确定的反应速率的酶促过程的每个动力学步骤和动力学中间体的性质的鉴定。这些研究对于了解ChuS和NOS在生理学和疾病中的作用以及设计有效和特异性的抑制剂至关重要。这些研究应该是一般感兴趣的学术界，工业界，制药和生物技术部门的酶进行氧化还原反应。

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.