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.
Highlights
To understand the role of the [4Fe-4S]2؉ cluster in controlling the activity of the Escherichia coli transcription factor FNR during changes in O2 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 [4Fe-4S]2ϩ Cluster of FNR-L28H Shows Increased Stability to O2 in Vitro—The observation that FNR-L28H, unlike WT FNR, is able to activate transcription under aerobic growth conditions [18, 21] suggested that the [4Fe-4S]2ϩ cluster in FNR-L28H might be less sensitive to O2 than WT-FNR
This minor effect on the absorption spectrum is in sharp contrast to what was observed previously for WT-FNR where a much larger decrease in the absorbance at 409 nm (ϳ50%) was observed after only 10 min of air exposure [13], suggesting that the [4Fe-4S]2ϩ cluster of FNR-L28H is more stable to air than WT-FNR
Summary
Plasmid and Strain Construction—Cysteine to serine substitutions at amino acid positions 20, 23, and 29 were created by site-directed mutagenesis [22]. For Mossbauer studies on purified FNR-L28H, 57Fe-enriched FNR-L28H was obtained in the same way except PK22 cells containing pPK1868 were grown aerobically in 57Fe-enriched glucose minimal medium [13], and the pooled fractions were concentrated over a 1-ml Biorex 70 column as described previously [11]. Whole Cell Mossbauer Spectroscopy—PK22 strains containing plasmid pPK823 (WT-FNR; Ref. 11) or pPK1868 (FNR-L28H) were grown in 57Fe-enriched glucose minimal medium [15] under either aerobic or anaerobic growth conditions, and FNR synthesis was induced by the addition of isopropyl-thio--D-galactoside for 1 h. O2-induced Degradation of the Fe-S Cluster—A 1-ml sample of anaerobically purified FNR-L28H protein (242 M) was placed in a 1.7-ml glass conical tube and stirred slowly in air at 18 °C as described previously [13] and assayed for sulfide and DNA binding as indicated below. Carbonic anhydrase, and cytochrome c protein standards (Sigma) were used to calibrate the column
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