铁氧还蛋白还原酶中[4Fe-4S]簇的重组及体外好氧-厌氧转录开关的验证。
Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro.
作者信息
Green J, Bennett B, Jordan P, Ralph E T, Thomson A J, Guest J R
机构信息
The Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, U.K.
出版信息
Biochem J. 1996 Jun 15;316 ( Pt 3)(Pt 3):887-92. doi: 10.1042/bj3160887.
Abstract
The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch.
摘要
大肠杆菌的FNR蛋白是一种氧化还原响应转录调节因子，可激活和抑制厌氧和好氧代谢所需的一系列基因。通过用亚铁离子、半胱氨酸和棕色固氮菌的NifS蛋白进行厌氧处理来重建野生型FNR，会导致每个FNR二聚体掺入两个[4Fe-4S]2+簇。重建后的FNR的紫外可见光谱在420nm处有一个宽吸收峰。这些簇在厌氧条件下是EPR沉默的，但通过空气的有限氧化会降解为[3Fe-4S]+，长时间暴露在空气中则会完全消失。CD光谱证实了FNR与铁硫簇的结合。与脱辅基FNR相比，[4Fe-4S]2+簇的掺入使位点特异性DNA结合增加了约7倍。体外厌氧转录激活和抑制同样依赖于铁硫簇的存在，并且其在有氧条件下的失活在体外证明了FNR介导的有氧-厌氧转录开关。
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