The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion.
Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations.
Jervis
Adrian J
AJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Crack
Jason C
JC
White
Gaye
G
Artymiuk
Peter J
PJ
Cheesman
Myles R
MR
Thomson
Andrew J
AJ
Le Brun
Nick E
NE
Green
Jeffrey
J
D000332:Aerobiosis; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D055550:Protein Stability; D012694:Serine / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D008024:Ligands; D014157:Transcription Factors; D012694:Serine; D010649:Phenylalanine; D010100:Oxygen