ABSTRACT
The ability of FNR to sense and respond to cellular O(2) levels depends on its [4Fe-4S](2+) cluster. In the presence of O(2), 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 approximately 2 Fe(2+) ions are released from the reaction of O(2) with the [4Fe-4S](2+) cluster. Fe(2+) 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 O(2)-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 O(2); a comparison of the in vitro and in vivo rates of conversion suggests that O(2)-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.
Subject(s)
Escherichia coli Proteins/physiology , Iron-Sulfur Proteins/physiology , Iron/metabolism , Oxygen/metabolism , DNA/metabolism , Escherichia coli Proteins/analysis , Iron-Sulfur Proteins/analysis , Kinetics , Promoter Regions, GeneticABSTRACT
The oxygen sensing ability of the transcription factor FNR depends on the presence of a [4Fe-4S]2+ cluster. In the presence of O2, conversion of the [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster inactivates FNR, but the fate of the [2Fe-2S]2+ cluster in cells grown under aerobic conditions is unknown. The present study shows that the predominant form of FNR in aerobic cells is apo-FNR (cluster-less FNR) indicating that the [2Fe-2S]2+ cluster, like the [4Fe-4S]2+ cluster, is not stable under these conditions. By quantifying the amount of [2Fe-2S]2+ cluster in 2Fe-FNR in vitro in the presence of various reductants and oxidants (GSH, DTT, cysteine, O2, hydrogen peroxide, and superoxide), we found that superoxide, a byproduct of aerobic metabolism, significantly destabilized the [2Fe-2S]2+ cluster. Mössbauer spectroscopy was used to monitor the effects of superoxide on 2Fe-FNR in vivo; under cellular conditions that favored superoxide production, we observed the disappearance of the signal representative of the [2Fe-2S]2+ cluster. We conclude that the [2Fe-2S]2+ cluster of FNR is labile to superoxide both in vitro and in vivo. This lability may explain the absence of the [2Fe-2S]2+ cluster form of FNR under aerobic growth conditions.
Subject(s)
Escherichia coli Proteins/chemistry , Iron-Sulfur Proteins/chemistry , Multienzyme Complexes/chemistry , Succinate Dehydrogenase/chemistry , Superoxides/chemistry , Transcription Factors/chemistry , Aerobiosis , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Iron/metabolism , Iron-Sulfur Proteins/isolation & purification , Iron-Sulfur Proteins/metabolism , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Oxidation-Reduction , Oxygen/metabolism , Solutions , Spectroscopy, Mossbauer , Succinate Dehydrogenase/isolation & purification , Succinate Dehydrogenase/metabolism , Sulfur/metabolism , Transcription Factors/isolation & purification , Transcription Factors/metabolismSubject(s)
Camphor 5-Monooxygenase/history , Electron Spin Resonance Spectroscopy/history , Iron-Sulfur Proteins/history , Camphor 5-Monooxygenase/chemistry , Crystallography/history , Crystallography/methods , Electron Spin Resonance Spectroscopy/methods , History, 20th Century , Iron-Sulfur Proteins/chemistry , Protein Conformation , Spin Trapping , United StatesABSTRACT
Fe-S clusters are key to the sensing and transcription functions of three transcription factors, FNR, IscR and SoxR. All three proteins were discovered in Escherichia coli but experimental data and bioinformatic predictions suggest that homologs of these proteins exist in other bacterial species, highlighting the widespread nature of Fe-S-dependent regulatory networks. In addition, the nearly ubiquitous citric acid cycle enzyme, aconitase, plays a role in translational regulation in E. coli and Bacillus subtilis when it loses its Fe-S cluster. Although these regulatory proteins have the common feature of containing an Fe-S cluster, they differ in the physiological signals that they respond to. Therefore, these regulatory factors provide insights into the chemical versatility of Fe-S clusters.
Subject(s)
Bacteria/genetics , Bacterial Proteins/physiology , Escherichia coli/genetics , Iron-Sulfur Proteins/physiology , Aconitate Hydratase/genetics , Aconitate Hydratase/metabolism , Bacteria/classification , Bacteria/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
An attempt is made to retrace, from personal experience, the discovery of redox-reactive non-heme iron in living matter, which turned out to occur in the form of iron-sulfur (Fe-S) clusters, and then to recount the immediate application of this knowledge in exploring the composition of the mitochondrial respiratory chain, and in the rather detailed description of the workings of its components and, for the purposes of the present volume, of succinate dehydrogenase. The relationship of these events to the general status of technology and the available methodology and instrumentation is considered in some detail, with the conclusion that there scarcely was a way that these discoveries could have been made earlier. It is then shown how methods, techniques and interpretations of results were developed and evolved during the applications that were made to a complex problem such as that of the composition, structure and functioning of succinate dehydrogenase. A tabulation of the most significant events--concerning specifically spectroscopy and its interpretations--in this development is given up to the year 2000.
