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Abstract
The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.
Highlights
Escherichia coli is a metabolically versatile chemoheterotroph, capable of growth on various substrates under various oxygen tensions
We report in vivo studies of D154A and I151A fumarate and nitrate reduction (FNR) variants that confirm the importance of FNR dimerization for transcriptional activity and showed that the rate of transcriptional response, dependent on [4Fe-4S] cluster incorporation or [4Fe-4S] into [2Fe-2S] conversion, is not significantly affected by, respectively, pre-loading of D154A FNR on to DNA or its inability to monomerize following O2 exposure
The activity of FNR is controlled by incorporation of an O2sensitive [4Fe-4S] cluster into FNR monomers resulting in the formation of FNR dimers that exhibit enhanced site-specific DNA binding [3,8,12]
Summary
Escherichia coli is a metabolically versatile chemoheterotroph, capable of growth on various substrates under various oxygen tensions. In E. coli, the fumarate and nitrate reduction (FNR) transcriptional regulator is responsible for sensing environmental levels of O2 and controlling the switch to anaerobic respiration [2,3,4,5]. In the absence of O2, monomeric (∼30 kDa) FNR acquires a [4Fe-4S]2+ cluster, triggering a conformational change at the dimerization interface that leads to the formation of homodimers (∼60 kDa) and site-specific DNA binding [10,11]. Cluster conversion results in a rearrangement of the dimer interface, leading to monomerization [10]. In this respect, E. coli and closely related FNR proteins are unique. Other members of the CRP-FNR family typically remain dimeric, irrespective of the presence of their analyte [15], showing that the conformational changes that switch the DNA affinity are not associated with a monomer–dimer equilibrium
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