基于轴向配位膜蛋白的胞外电子转移活体调控及机制解析

Extracellular respiration is a novel microbial metabolism, which involves electron transfer to or from extracellular substrates, termed as extracellular electron transfer. Extracellular respiration shows environmental significance and potential application in bioremediation, wastewater treatment, bioenergy recovery and so on. However, the low electron transfer efficiency is one of the major limitions to the practical application. Currently, great efforts have been paid to greatly improve the extracellular electron tranfser efficiency. Membrane-bound redox proteins are the key components of the extracellular electron transfer chain. It has been found that the electron tranfer process of these redox proteins could be controlled through axial ligation. However, previous studies mainly focused on the electron tranfer control of puried proteins and less attention has been paid to the in vivo redox proteins. In this project, based on the characteristics of extracellular electron transfer, we propose to control the redox process of the living redox proteins through axial ligation. We will systemically study the electrochemical properties of living porteins in the presence of different types of axial ligants. We will also probe the effects of the axial ligation on the microbial iron reduction, microbial electrode redcution as well as catalytical oxygen reduction, and reveal the intrinsic mechanism of in vivo control of redox proteins. The results will provide new clues for understanding the extracellular electront transfer process, and may also provide technical support for realizing the practial application of extracellular respiration in environemts.

微生物胞外呼吸是近年来发现的新型能量代谢方式，其核心科学问题是胞外电子传递过程，即微生物氧化产生的电子如何穿过非导电的细胞膜/壁传递至胞外固态受体。细胞色素c(c-Cyts)是胞外电子传递链的关键组分，但对其介导电子转移的微观机制与调控手段却缺乏研究。轴向配位C-Cyts技术，为揭示和调控胞外电子传递微观过程提供了可能。但是，该方法目前仅限于纯化蛋白，活体蛋白却少受关注。本项目从c-Cyts的结构特征与配位特点入手，提出基于轴向配位的活体c-Cyts调控新方法，系统研究各类轴向配体配位c-Cyts的电化学行为，探讨其对铁还原、电极还原和氧还原催化过程的影响，综合采用生物电化学与现代光谱技术，阐明活体c-Cyts介导胞外电子转移的分子机制，设计合成高效促进电子转移的固体轴向配体，为深入理解微生物胞外电子转移途径提供理论基础，为拓展其在环境修复和生物能源利用的应用提供技术支撑。

本项目以活体微生物（Geobacter和Shewanella）细胞色素c（c-Cyts）为研究对象，从c-Cyts的结构特征与配位特点入手，探讨轴向配位取代对c-Cyts氧化还原能力和微生物胞外电子转移的影响，揭示轴向配位活体c-Cyts的分子机制；利用活体c-Cyts的轴向配位特性结合生物电化学和现代光谱学技术，研究胞外电子转移机制和调控技术。项目重点研究了活体膜蛋白(c-Cyts)在不同电极界面的氧化还原行为；轴向配位作用对活体膜蛋白(c-Cyts)氧化还原行为的影响；建立了基于紫外可见光/拉曼/电化学表面等离子体共振（EC-SPR-OFS）等研究配体轴向配位c-Cyts过程以及电子传递过程机制的方法；利用量子化学计算方法在理论上预测了轴向配位c-Cyts对其胞外电子过程的影响及机制；并且设计合成了基于轴向配位的N掺杂石墨烯和咪唑功能化石墨烯等胞外电子转移促进剂。通过本项目的实施，基本明确了轴向配位取代对活体细胞色素c氧化还原能力的影响，提出了基于轴向配位的活体c-Cyts调控新方法，揭示了活体c-Cyts介导的胞外电子转移微观机制，设计了合成可高效促进电子转移的固体界面（包含轴向配体），为拓展微生物胞外呼吸在环境修复和生物能源利用的实际应用提供技术支撑。实际完成SCI论文14篇，发明专利2项，联合培养硕士、博士生4名，圆满完成预期任务。