奥奈达希瓦氏菌CctA介导周质甲基橙还原的电子传递机理

Electron transport mechanism of periplasmic methyl orange reduction mediated by CctA in Shewanella oneidensis

摘要:

电活性微生物奥奈达希瓦氏菌的胞外电子传递（extracellular electron transfer，EET）在污染物降解、环境修复、生物电化学传感、能源利用等方面具有广泛的应用潜力；四血红素细胞色素CctA （small tetraheme cytochrome）是希瓦氏菌周质空间中最丰富的蛋白质之一，能够参与多种氧化还原过程，但目前对CctA在EET中的行为和机理认识仍然有限。【目的】研究阐明CctA蛋白在希瓦氏菌模式菌株MR-1周质空间以偶氮染料作为电子受体的EET中的作用，补充和拓展希瓦氏菌的厌氧呼吸产能机制。【方法】以周质还原型偶氮染料甲基橙（methyl orange，MO）作为电子受体，在mteal reduction （Mtr）蛋白缺失菌株Δmtr中研究MO的周质还原特点，并通过基因敲除和回补表达研究CctA蛋白在周质电子传递中的作用。【结果】在缺失Mtr通道的情况下，细胞色素CctA可以介导周质空间的电子传递而还原MO。重组表达CctA在低水平时，MO在周质空间中的还原速率与其表达水平呈正相关，更高水平的CctA表达无助于进一步提高MO的还原速率。蛋白膜伏安结果展示了CctA与周质空间内其他高电位氧化还原蛋白的显著区别，可能参与构成一条低电位的MO还原通道。【结论】从分子动力学层面揭示了CctA在周质MO还原中的独特电子传递行为，为进一步推进对细菌周质电子传递机制的理解，以及通过合成生物学设计或改造胞外氧化还原系统、强化生物电化学在污染物降解中的应用提供了重要信息。

关键词:细胞色素c;希瓦氏菌;周质蛋白;甲基橙;胞外电子传递

Abstract:

Extracellular electron transfer (EET) of the electrochemically active microbe, Shewanella oneidensis, has promising prospects of application in pollutant degradation, environmental remediation, bioelectrochemical sensing, and energy utilization. The small tetrahaem cytochrome (CctA) is one of the most abundant proteins in the periplasmic space of S. oneidensis. Although CctA is involved in multiple redox processes, the knowledge on the behavior and mechanism of CctA in EET is limited. [Objective] To investigate the role of CctA in the EET with azo dyes as electron acceptors in the periplasmic space of the model strain S. oneidensis MR-1, and further enrich the knowledge about the mechanism of anaerobic respiration of S. oneidensis. [Methods] We took methyl orange (MO) as the electron acceptor to explore the characteristics of periplasmic MO reduction in ∆mtr (mtr-deleted strain) and investigate the role of CctA in EET by gene knockout and complementation. [Results] In the absence of Mtr complexes, CctA mediates the electron transfer for the periplasmic MO reduction. The reduction rate of MO had a positive correlation with the expression level of recombinant CctA at low concentrations, while higher levels of recombinant CctA did not further improve the reduction rate. The results of protein film voltammetry (PFV) distinguished CctA significantly from high-potential redox proteins in the periplasmic space, suggesting its involvement in a low-potential MO reduction pathway. [Conclusion] We uncovered the unique electron transfer behavior of CctA in periplasmic MO reduction from molecular dynamics, bringing valuable information for understanding EET, designing or modifying extracellular redox systems by means of synthetic biology, and facilitating the application of bioelectrochemistry in pollutant degradation.

Key words:cytochrome c;Shewanella;periplasmic proteins;methyl orange;extracellular electron transfer (EET)