title: Mass spectrometric identification of intermediates in the O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion in FNR.
abstract: The iron-sulfur cluster containing protein Fumarate and Nitrate Reduction (FNR) is the master regulator for the switch between anaerobic and aerobic respiration inEscherichia coliand many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O2that leads to conversion to a [2Fe-2S] form with loss of high-affinity DNA binding. Here, we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster and persulfide-coordinated [2Fe-2S] clusters [[2Fe-2S](S)n, wheren= 1 or 2]. Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion and not as a subsequent, secondary reaction to generate [2Fe-2S](S)nspecies. This methodology shows great potential for broad application to studies of protein cofactor-small molecule interactions.
authors.affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom.
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, United Kingdom n.le-brun@uea.ac.uk.
authors.forename: Nick E
authors.identifier: 0000-0001-9780-4061
authors.initials: NE
authors.lastname: Le Brun
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005650:Fumarates / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009566:Nitrates / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology*; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D005650:Fumarates; D007506:Iron-Sulfur Proteins; D009566:Nitrates; D013440:Sulfides; D007501:Iron; D010100:Oxygen
keywords: DNA regulation; iron-sulfur; mass spectrometry; oxygen sensor