title: Influence of association state and DNA binding on the O₂-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator.
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.
authors.affiliation: *Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.affiliation: †Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: Melanie R
authors.initials: MR
authors.lastname: Stapleton
authors.affiliation: †Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.affiliation: *Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
authors.affiliation: *Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.
authors.forename: Nick E
authors.initials: NE
authors.lastname: Le Brun
mesh_terms: D019943:Amino Acid Substitution; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D010100:Oxygen