PMID:10692418
| Citation |
Bates, DM, Popescu, CV, Khoroshilova, N, Vogt, K, Beinert, H, Münck, E and Kiley, PJ (2000) Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe-4S](2+) cluster to oxygen. J. Biol. Chem. 275:6234-40 |
|---|---|
| Abstract |
To understand the role of the [4Fe-4S](2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions. |
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| Keywords |
Aerobiosis; Anaerobiosis; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; Dimerization; Escherichia coli/metabolism; Escherichia coli Proteins; Iron-Sulfur Proteins/chemistry; Iron-Sulfur Proteins/genetics; Mutation; Oxygen/pharmacology; Spectrophotometry; Spectroscopy, Mossbauer; Sulfides/analysis; Transcription Factors/chemistry; Transcription Factors/genetics |
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