The Electrochemistry of Diheme Cytochrome c Peroxidases

描述(由申请人提供)：这项研究使用蛋白质膜伏安法(PFV)的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。细菌生物体和所有生物体一样，通过使用特定的酶来破坏有毒的过氧化氢。就细菌而言，二铁血红素过氧化物酶(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，因为它保护有机体免受氧化条件的影响，例如宿主的自然防御系统产生的氧化条件。在这项计划中，我们将研究细菌CCP中电子转移和过氧化氢还原的机制。有趣的是，一些CCP在完全氧化后很容易失活；另一些则没有这一特性。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存在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反应活性的分子细节，以及过氧化物酶的氧化还原状态与激活和失活的关系。我们假设CCP的初级序列中有少量的决定因素，表明激活需要。我们将(1)测量野生型亚硝酸单胞菌欧洲酶的还原电位和电化学性质，它不需要氧化还原连接的激活；(2)研究脱氮副球藻CCP内的激活/失活反应，这是已知需要激活的；(3)产生一个超表达系统，这将使我们能够从事定点突变研究；以及(4)表征一个尚未在文献中描述的新的三血红素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，详细了解生物如何防御过氧化氢等活性氧物种。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，为了解它们的生化途径提供新的见解。

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.