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.
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.
Bates
D M
DM
Department of Biomolecular Chemistry, Medical School, University of Wisconsin, Madison, Wisconsin 53706, USA.
Popescu
C V
CV
Khoroshilova
N
N
Vogt
K
K
Beinert
H
H
Münck
E
E
Kiley
P J
PJ
D000332:Aerobiosis; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology*; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013440:Sulfides / Q000032:analysis; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D013440:Sulfides; D014157:Transcription Factors; D010100:Oxygen