类希瓦氏菌WH16-1中基于FixABC的新型铬还原电子传递机制

铬还原菌能将高毒性的Cr(VI)还原为低毒性的Cr(III)，具有自身和环境解毒意义。目前鉴定的细菌有氧和无氧铬还原酶主要为黄素蛋白和细胞色素c蛋白，新型铬还原酶有待进一步发掘。近期，申请人发现类希瓦氏菌WH16-1中的铁氧还原蛋白(Fd)和细胞色素bd蛋白(Cytbd)分别与有氧和无氧铬还原相关，而Fd和Cytbd的潜在电子供体蛋白FixABC受Cr(VI)诱导上调。本项目拟通过转录分析明确fixABC、fd、cytbd对Cr(VI)胁迫的响应；通过基因敲除、互补及异源超表达，确定fixABC、fd、cytbd在体内对Cr(VI)还原的贡献；通过表达纯化、体外酶活检测FixABC、Fd、Cytbd的活性；通过电化学方法表征和体外电子传递模拟，验证推测的电子传递通路。最终，阐明有氧条件下FixABC-Fd和无氧条件下FixABC-Cytbd的Cr(VI)还原机制，为铬污染修复提供理论基础。

Chromate reducing bacteria reduce high toxic Cr(VI) to less toxic Cr(III), which can participate in autologous or environmental detoxification of Cr(VI). Up to now, the reported aerobic and anaerobic Cr(VI) reductases were primarily flavin proteins and cytochrome c proteins, respectively. The novel chromium reduction electron transfer pathways need to be further explored. Recently, we found that Ferredoxin (Fd) and cytochrome bd (Cytbd) were associated with aerobic and anaerobic Cr(VI) reduction in Alishewanella sp. WH16-1, respectively. The electron transport protein FixABC, a potential electron donor of Fd and Cytbd, was up-regulated under Cr(VI) stress. Based on these discoveries, this application will detect the response of fixABC, fd and cytbd to Cr (VI) stress by transcription analysis. The contribution of fixABC, fd and cytbd to Cr (VI) reduction in vivo will be determined by gene knockout, complementation and heterologous overexpression. Moreover, the activity of FixABC, Fd and Cytbd will be measured by expression, purification and in vitro enzyme activity tests. In addition, the putative electron transfer pathway will be confirmed by electrochemical characterization and in vitro electron transfer simulation. Finally, the novel Cr(VI) reduction molecular mechanism of FixABC-Fd and FixABC-Cytbd under aerobic and anaerobic conditions will be identified, respectively. It will provide a theoretical basis for the remediation of chromium contamination.