| In Escherichia coli, the global transcriptional regulator FNR senses and responds to oxygen in order to promote anaerobic metabolism. FNR is active as a transcriptional regulator under anaerobic conditions, in which each FNR molecule contains a [4Fe-4S]2+ cluster that is required for activity. The presence of the [4Fe-4S]2+ cluster promotes homo-dimerization of FNR molecules to produce the form (called 4Fe-FNR) that is active for DNA binding and transcriptional regulation. In the presence of oxygen, each [4Fe-4S]2+ cluster is converted to a [2Fe-2S] 2+ cluster, thereby inactivating FNR as a transcriptional regulator. The fate of the [2Fe-2S]2+ cluster form of FNR (called 2Fe-FNR) in aerobic cells was previously unknown.; A comprehensive description of the methods used in the preparation and analysis of the oxygen-sensitive protein 4Fe-FNR was prepared. In order to understand the role of 2Fe-FNR in cells, a related method was developed for 2Fe-FNR purification. The use of this method to purify FNR from aerobically grown cells demonstrated that the predominant form of FNR under aerobic growth conditions is apo-FNR, which contains no Fe-S cluster. In order to identify the factor that converts 2Fe-FNR into apo-FNR in cells, purified 2Fe-FNR was subjected to stability analyses in the presence of various redox reagents, which demonstrated that superoxide specifically destabilizes the [2Fe-2S] 2+ cluster of FNR. Whole cell Mossbauer spectroscopy was then used to demonstrate that 2Fe-FNR is converted to apo-FNR in vivo in the presence of superoxide. The discovery of the superoxide-lability of the 2Fe-FNR form enhances our understanding of the mechanism by which FNR is maintained in an inactive state under aerobic growth conditions.; As a first step toward a comprehensive model of oxygen-sensing by FNR, the reaction of purified 4Fe-FNR with oxygen was analyzed. Fe2+ ions were identified as a product of the reaction, and the release of Fe 2+ was used to monitor the progress of the reaction in varying oxygen concentrations. This process was compared with in vivo FNR inactivation by monitoring expression from FNR-regulated promoters. The results suggest that the oxygen-induced conversion of 4Fe-FNR to 2Fe-FNR is sufficient to account for the inactivation of FNR observed in vivo. |
