Observation of the Iron-Sulfur Cluster in Escherichia coli Biotin Synthase by Nanoflow Electrospray Mass Spectrometry
| Autor: | N M Shaw, J E Baldwin, Peter L. Roach, K S Hewitson, Carol V. Robinson, Helena Hernández |
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| Rok vydání: | 2001 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Chromatography biology Molecular mass Stereochemistry Iron Electrospray ionization Dimer Iron–sulfur cluster Biotin synthase Buffers Mass spectrometry Tandem mass spectrometry Analytical Chemistry chemistry.chemical_compound chemistry Ionic strength Sulfurtransferases Escherichia coli biology.protein Sulfur |
| Zdroj: | Analytical Chemistry. 73:4154-4161 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac0102664 |
| Popis: | 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+. |
| Databáze: | OpenAIRE |
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