基于活菌外膜蛋白氧化还原状态的胞外电子传递动力学

The c-type cytochromes play a significant role on the extracellular electron transfer (EET). In vitro studies of cytochromes were used to illustrate their EET properties previously. As a large discrepancy has been reported between purified proteins and protein complexes in intact cells, in vivo cytochrome-based EET kinetic study will be valuable for a comprehensive understanding of the EET mechanism. On a basis of the development of a time-resolved spectrometer, this project will focus on the EET kinetic study in terms of the in vivo outer-membrane proteins. In the project, the iron reduction bacterium Shewanella oneidensis MR-1 will be chosen as a model strain, and the redox status as well as the rapid redox process of the outer-membrane cytochromes in intact cells will be studied under different initial concentrations and/or types of electron donors and acceptors. This study is intended to elucidate the relationship between the cytochrome reduction rate and the following values: the rate constant of the elementary processes (k), the rates of the elementary processes (r), the real-time redox status of cytochrome, and the concentrations of electron donors and acceptors, and to illuminate the quantitative relationship of the electron transfer rate and the formal redox potential between cytochromes and electron acceptors. We will establish a kinetic model that describing the cytochrome reduction rate and a model that describing the dynamic equilibrium of cytochrome redox status to quantitatively elucidate the kinetics of EET based on the redox status of in vivo outer-membrane proteins. The study will provide a substantial implication for improving the fundamental understanding of the biogeochemical processes driven by EET.

微生物外膜细胞色素c(蛋白)是胞外电子传递的引擎。以往主要采用离体蛋白来研究其胞外电子传递特性，难以真实反映活菌的状态。因此，基于活菌蛋白的胞外电子传递动力学研究具有重要意义。在已建立的活菌蛋白状态10毫秒级测试方法基础上，选取希瓦斯菌MR-1为模式菌，系统研究不同电子供体和受体的类型和初始浓度状况下，活菌蛋白的氧化还原平衡状态，以及活菌蛋白被电子供体快速还原、被电子受体快速氧化的基元过程动力学。阐明细胞色素c总的还原速率与各基元过程动力学常数k值、基元过程速率r值、细胞色素c的实时氧化还原状态、供体和受体浓度之间的关联，以及电子传递速率和细胞色素c与电子受体的实际电势差之间的定量关系。构建描述活菌细胞色素c总还原速率的动力学模型，以及描述细胞色素c氧化还原动态平衡的模型。从而，在活菌外膜蛋白层面定量阐明胞外电子传递动力学机制。为深入理解胞外电子传递驱动的生物地球化学过程提供科学依据。

微生物外膜细胞色素c(蛋白)是胞外电子传递的引擎。以往主要采用离体蛋白来研究其胞外电子传递特性，难以真实反映活菌的状态。因此，基于活菌蛋白的胞外电子传递动力学研究具有重要意义。通过本项目的实施，搭建了漫透射光谱电化学联用装置，原位测试了活菌细胞色素c及电子受体的氧化态和还原态含量变化，并同时在线监测了反应体系的氧化还原电位值，从而实现了实测电位与各反应物计算电位之间相互关系的系统研究。基于此，本项目从动力学和热力学角度解析了细胞色素c和电子受体两者实时氧化还原状态的依存关系；阐明了细胞色素c在溶液中充当电子穿梭体的动力学机制，首次证实了细胞色素c可作为穿梭体介导微生物与Cr之间的电子转移；在此基础上，发现了穿梭体加速生物膜形成促进电子传递的胞外呼吸机制，为后续研究奠定了基础。本项目为深入理解胞外电子传递驱动的生物地球化学过程提供了科学依据。