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: 7492994
• 负责人: SEAN J ELLIOTT
• 金额: $ 20.12万
• 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/7492994
• 关键词: Active Sites; Bacteria; Base Sequence; Biochemical; Biochemical Pathway; Biology; Catalysis; Characteristics; Chemistry; Class; Complement; Condition; 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; Numbers; Organism; Oxidation-Reduction; Paracoccus denitrificans; Peroxidase; Peroxidases; Peroxides; Positioning Attribute; Property; Protein Overexpression; Proteins; Protons; Range; 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; 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）：本研究使用蛋白质膜伏安法（PFV）的电化学工具唯一地报告了细菌CCP酶的催化化学，氧化还原特性以及激活和失活反应。像所有生物一样，细菌生物通过使用特定酶破坏了过氧化有毒的氢。就细菌而言，二甲乳酶过氧化物酶（CCP）从细胞色素c中获取电子，并使用它们将过氧化氢降低到水中。这种反应对于微生物的生存至关重要，因为它可以捍卫生物体免受氧化条件的影响，例如宿主的自然防御系统产生的条件。在此提案中，我们将研究来自细菌CCP的电子转移和过氧化物降低的机制。有趣的是，有些CCP完全氧化后很容易被灭活。其他人没有这种特征。细菌CCP在序列和结构上似乎是同源的，这使得过氧化物酶在吸引人中引起这种差异的分子原因。我们研究的远距离目标是了解确定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有关。我们假设CCP的主要序列中有少数决定因素，表明激活的要求。我们将（1）测量野生型硝基瘤欧洲酶的还原电位和电化学特性，而欧罗波亚酶不需要氧化还原连接的激活； （2）研究denitrificans的CCP内的激活/失活反应，已知需要激活； （3）生成Shewanella Oneidensis酶的过表达系统，这将使我们能够从事定向的诱变研究； （4）表征了文献中尚未描述的新型Triheme CCP的新型子类。生物医学影响：提出的实验将通过了解生物学如何防御活性氧（例如过氧化氢）的详细理解，通过了解氧化还原化学与酶机制之间的相互作用。此外，我们对Triheme CCP的研究将阐明CCP机制，该机械是诸如肠沙门氏菌和耶尔森氏菌病原体所独有的，为其生化途径提供了新的见解。