Observation of the iron-sulfur cluster in Escherichia coli biotin synthaseby nanoflow electrospray mass spectrometry

Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry. From solution conditions in which the protei n is in its native state, a distribution of monomeric, dimeric, and tetrame ric species was observed. The distribution of these species was sensitive t o 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] cl uster with a mass difference of 175.3 Da from the apomonomer with one disul fide bond. Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur clust er in both positive and negative ion spectra. Additionally, observation of a series of charge states assigned to the apodimer indicated that binding o f the iron-sulfur cluster was not required to maintain the dimer. Binding o f Cu2+ to biotin synthase was also observed; in the presence of excess chel ating 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 ch arge state containing the cluster at m/z 2416.9 was isolated, and collision -induced dissociation resulted in sequential loss of sulfur and retention o f Fe3+.