Title

Reconstitution of the [4Fe-4S] Cluster in FNR and Demonstration of the Aerobic–Anaerobic Transcription Switch in vitro

Document Type

Article

Language

eng

Format of Original

6 p.

Publication Date

6-15-1996

Publisher

Portland Press Limited

Source Publication

Biochemical Journal

Source ISSN

0264-6021

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.

Comments

Biochemical Journal, Vol. 316, No. 3 (June 15, 1996): 887-892. Permalink.

Brian Bennett was affiliated with the University of East Anglia at the time of publication.