The Electrochemistry of Diheme Cytochrome c Peroxidases

二血红素细胞色素 c 过氧化物酶的电化学

• 批准号: 7144879
• 负责人: SEAN J ELLIOTT
• 金额: $ 22.31万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7144879
• 关键词: aerobic bacteria; bacterial genetics; bacterial proteins; biofilm; chemical kinetics; cytochrome c peroxidase; electrical potential; electrochemistry; electron spin resonance spectroscopy; electron transport; enzyme activity; gene induction /repression; heme; hemoprotein; oxidation reduction reaction; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant): This study uses the electrochemical tool of protein film voltammetry (PFV) to uniquely report upon the catalytic chemistry, redox properties, and activation and deactivation reactions of bacterial CCP enzymes. Bacterial organisms, like all organisms, destroy toxic hydrogen peroxide by the use of specific enzymes. In the case of bacteria, diheme peroxidases (CCPs) take electrons from cytochrome c and use them to reduce hydrogen peroxide to water. This reaction is crucial to survival for the microbes, as it defends the organism against oxidizing conditions, such as those engendered by a host's natural defense systems. In this proposal, we will study the mechanisms of electron transfer and peroxide reduction in CCPs from bacteria. Interestingly some CCPs become easily inactivated when they are fully oxidized; others do not have this trait. Bacterial CCPs seem to be homologous in sequence and structure, which makes the molecular cause for this difference amongst the peroxidases intriguing. The long-range goals of our study are to understand the molecular details that determine if a CCP reactivity, and how the redox state of a peroxidase relates to activation and inactivation. We hypothesize that there are a small number of determinants in the primary sequence of CCPs, indicating the requirements for activation. We will (1) measure the reduction potentials and electrochemical characteristics of the wild type Nitrosomonas europaea enzyme, which does not require redox-linked activation; (2) study the activation/deactivation reaction within the CCP from Paracoccus denitrificans, which is known to require activation; (3) generate an overexpression system of the Shewanella oneidensis enzyme, that will allow us to engage in site-directed mutagenesis studies; and (4) characterize a novel sub-class of triheme CCPs that have yet to be described in the literature. Biomedical impact: The proposed experiments will yield a detailed understanding of how Biology defends itself against reactive oxygen species such as hydrogen peroxide, by understanding the interplay between redox chemistry and enzyme mechanism. Further, our study of triheme CCPs will elucidate the CCP machinery which is unique to pathogens such as Salmonella enterica and Yersinia pestis, providing new insights into their biochemical pathways.

描述（由申请人提供）：本研究使用蛋白质膜伏安法 (PFV) 电化学工具来独特地报告细菌 CCP 酶的催化化学、氧化还原特性以及激活和失活反应。与所有生物体一样，细菌生物体通过使用特定的酶来破坏有毒的过氧化氢。就细菌而言，二血红素过氧化物酶 (CCP) 从细胞色素 c 获取电子，并利用它们将过氧化氢还原为水。这种反应对于微生物的生存至关重要，因为它可以保护生物体免受氧化条件的影响，例如宿主自然防御系统产生的氧化条件。在本提案中，我们将研究细菌 CCP 中电子转移和过氧化物还原的机制。有趣的是，一些 CCP 在完全氧化后很容易失活。其他人不具备这种特质。细菌 CCP 在序列和结构上似乎是同源的，这使得过氧化物酶之间这种差异的分子原因很有趣。我们研究的长期目标是了解决定 CCP 反应性的分子细节，以及过氧化物酶的氧化还原状态如何与激活和失活相关。我们假设 CCP 的一级序列中存在少量决定簇，表明了激活的要求。我们将（1）测量野生型亚硝化单胞菌酶的还原电位和电化学特性，该酶不需要氧化还原连接的激活； (2) 研究脱氮副球菌 CCP 内的激活/失活反应，已知该反应需要激活； (3) 生成 Shewanella oneidensis 酶的过表达系统，这将使我们能够进行定点诱变研究； (4)描述了尚未在文献中描述的三血素CCP的新亚类的特征。生物医学影响：通过了解氧化还原化学和酶机制之间的相互作用，拟议的实验将详细了解生物学如何防御过氧化氢等活性氧。此外，我们对三血红蛋白 CCP 的研究将阐明肠沙门氏菌和鼠疫耶尔森氏菌等病原体特有的 CCP 机制，为它们的生化途径提供新的见解。