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Journal of Bacteriology, December 2004, p. 8018-8025, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8018-8025.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Kinetic Analysis of the Oxidative Conversion of the [4Fe-4S]2+ Cluster of FNR to a [2Fe-2S]2+ Cluster

Victoria R. Sutton,1 Erin L. Mettert,2 Helmut Beinert,3 and Patricia J. Kiley2*

Program in Cellular & Molecular Biology,1 Department of Biomolecular Chemistry,2 Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin3

Received 20 May 2004/ Accepted 31 August 2004

The ability of FNR to sense and respond to cellular O2 levels depends on its [4Fe-4S]2+ cluster. In the presence of O2, the [4Fe-4S]2+ cluster is converted to a [2Fe-2S]2+ cluster, which inactivates FNR as a transcriptional regulator. In this study, we demonstrate that ~2 Fe2+ ions are released from the reaction of O2 with the [4Fe-4S]2+ cluster. Fe2+ release was then used as an assay of reaction progress to investigate the rate of [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion in vitro. We also found that there was no detectable difference in the rate of O2-induced cluster conversion for FNR free in solution compared to its DNA-bound form. In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O2; a comparison of the in vitro and in vivo rates of conversion suggests that O2-induced cluster conversion is sufficient to explain FNR inactivation in cells. FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions.

* Corresponding author. Mailing address: 1300 University Ave., Rm. 574, Madison, WI 53706. Phone: (608) 262-6632. Fax: (608) 262-5253. E-mail: pjkiley{at}wisc.edu.

Journal of Bacteriology, December 2004, p. 8018-8025, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8018-8025.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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