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研究血红素酶的动力学机制，即使用血红素作为辅助因子的酶。血红素酶在生物降解、性激素合成、生物修复、木质素降解等方面发挥着重要作用。我们提出了两种血红素酶在细菌（ChuS和NOS）和人类（NOS）中参与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，主要分布在环境细菌和致病菌（志贺氏菌、大肠杆菌等）中，使这些细菌能够利用血红素作为铁的来源进行生长。在人类和动物中不存在ChuS和ChuS同源物。因此，这种酶可能成为对抗致病性细菌感染的额外目标。一氧化氮是合成气态自由基一氧化氮（NO）的血红素酶，在人体内具有信使和细胞毒分子的双重功能。一氧化氮参与人体生理学（血压控制、免疫反应、神经通讯）和动脉粥样硬化、神经退行性疾病、糖尿病和癌症等疾病的许多方面。在细菌的一个子集中也发现了nos，在那里它们表现出惊人的多样化但关键的功能。ChuS和NOS都是血红素酶，必须激活分子氧和/或过氧化氢才能发挥其功能，其中涉及氧化反应。我们建议通过使用各种生物物理技术来破译NOS和ChuS进行底物氧化的详细动力学机制，这些技术将允许确定酶促过程中每个动力学步骤的反应速率和鉴定动力学中间体的性质。这些研究对于了解ChuS和NOS在生理和疾病中的作用以及设计有效和特异性抑制剂至关重要。这些研究应该引起学术界、工业界、制药和生物技术部门对酶进行氧化还原反应感兴趣的人的普遍兴趣。

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.