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: 7937401
• 负责人: SEAN J ELLIOTT
• 金额: $ 14.26万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937401
• 关键词: Active Sites; Bacteria; Base Sequence; Biochemical; Biochemical Pathway; Biology; Catalysis; Characteristics; Chemistry; Complement; Cytochrome c Peroxidase; Drug Metabolic Detoxication; Electrochemistry; Electron Transport; Electrons; Enzymes; Escherichia coli; Film; Generations; Goals; Heme; Heme Group; Hydrogen Peroxide; Kinetics; Knock-out; Ligands; Link; Lipids; Literature; Measures; Metals; Methods; Microbe; Modification; Molecular; Molecular Conformation; Nitrosomonas europaea; Organism; Oxidation-Reduction; Paracoccus denitrificans; Peroxidases; Peroxides; Positioning Attribute; Property; Proteins; Protons; Reaction; Reactive Oxygen Species; Reagent; Recombinants; Reporting; Research Personnel; Salmonella enterica; Scanning; Series; Shewanella; Site; Site-Directed Mutagenesis; Spectrum Analysis; Structure; System; Variant; Water; Yersinia pestis; base; cytochrome c; diheme cytochrome c; enzyme mechanism; genetic manipulation; insight; mutant; novel; overexpression; pathogen; programs; research study; tool; trait

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）：本研究使用蛋白膜伏安法（PFV）的电化学工具，以独特的方式报告细菌CCP酶的催化化学、氧化还原特性以及活化和失活反应。细菌有机体，像所有的有机体一样，通过使用特定的酶来破坏有毒的过氧化氢。在细菌的情况下，二血红素过氧化物酶（CCP）从细胞色素c中获取电子，并利用它们将过氧化氢还原为水。这种反应对微生物的生存至关重要，因为它保护生物体免受氧化条件的影响，例如宿主的天然防御系统所产生的氧化条件。在本论文中，我们将研究细菌CCP的电子转移和过氧化物还原机制。有趣的是，一些CCP在完全氧化时很容易失活;其他CCP则没有这种特性。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耐人寻味。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，有一个小数目的决定因素在初级序列的CCP，表明激活的要求。我们将（1）测量野生型欧洲亚硝化单胞菌酶的还原电位和电化学特征，该酶不需要氧化还原相关的活化;（2）研究脱氮副球菌CCP内的活化/失活反应，已知该反应需要活化;（3）产生希瓦氏菌（Shewanella oneidensis）酶的过表达系统，这将允许我们进行定点诱变研究;和（4）表征了一种新的三血红素CCP亚类，其尚未在文献中描述。生物医学影响：拟议的实验将通过了解氧化还原化学和酶机制之间的相互作用，详细了解生物学如何保护自己免受过氧化氢等活性氧的侵害。此外，我们对triheme CCP的研究将阐明CCP机制，这是肠道沙门氏菌和鼠疫耶尔森氏菌等病原体所独有的，为它们的生化途径提供了新的见解。