title: Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry.
abstract: Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry. From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed. The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer. The masses of the monomeric species were consistent with the presence of a [2Fe-2S] cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond. Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra. Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer. Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact. Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment. A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+.
authors.affiliation: Oxford Centre for Molecular Sciences, Oxford University, New Chemistry Laboratory, UK.
authors.forename: H
authors.initials: H
authors.lastname: Hernández
authors.forename: K S
authors.initials: KS
authors.lastname: Hewitson
authors.forename: P
authors.initials: P
authors.lastname: Roach
authors.forename: N M
authors.initials: NM
authors.lastname: Shaw
authors.forename: J E
authors.initials: JE
authors.lastname: Baldwin
authors.forename: C V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D002021:Buffers; D004926:Escherichia coli / Q000201:enzymology*; D007501:Iron / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013455:Sulfur / Q000737:chemistry*; D013466:Sulfurtransferases / Q000737:chemistry*
chemical_list: D002021:Buffers; D013455:Sulfur; D007501:Iron; D013466:Sulfurtransferases; C057985:biotin synthetase