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）：本研究利用蛋白膜伏安法（PFV）的电化学工具，对细菌CCP酶的催化化学、氧化还原性质、活化和失活反应进行了独特的报道。细菌和所有生物一样，通过使用特定的酶来破坏有毒的过氧化氢。以细菌为例，二血红素过氧化物酶（CCPs）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侵害，比如由宿主自然防御系统产生的氧化条件。在这个提议中，我们将研究细菌ccp的电子转移和过氧化还原机制。有趣的是，一些ccp在完全氧化时很容易失活；其他人则没有这种特质。细菌ccp在序列和结构上似乎是同源的，这使得这种过氧化物酶之间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活化和失活相关。我们假设在ccp的初级序列中有少量的决定因子，表明激活的要求。我们将(1)测量不需要氧化还原连接激活的野生型europaea亚硝化单胞菌酶的还原电位和电化学特性；(2)研究反硝化副球菌在CCP内的活化/失活反应，该反应已知需要活化；(3)生成一种希瓦氏菌酶的过表达系统，这将使我们能够从事定点诱变研究；(4)表征尚未在文献中描述的新型三血红素ccp亚类。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活性氧（如过氧化氢）的侵害。此外，我们对三血红素CCP的研究将阐明肠道沙门氏菌和鼠疫耶尔森菌等病原体特有的CCP机制，为其生化途径提供新的见解。