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 Fe2+ ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe3+ and 2 S2- 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.