Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mas s spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduce d into the ESI source under nondenaturing conditions in order to observe in tact metal-containing protein ions. The stoichiometry and oxidation state o f the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum, Mononuclear metal-containing proteins and [4Fe-4S] cente rs are very stable and yield the molecular ion with little or no fragmentat ion. proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecu lar ion is more abundant than the fragment peaks. [3Fe-4S]-containing prote ins are the least stable of the species investigated, yielding abundant pea ks corresponding to the loss of one to four sulfur atoms in addition to a p eak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-conta ining ions than obtained in positive ion mode. ESI analysis of the same pro teins under denaturing conditions yields mass spectra of the apo form of th e proteins. Disulfide bonds are observed in the apoprotein mass spectra tha t are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal ce nter. In addition, inorganic sulfide is found to incorporate itself into th e apoprotein by forming sulfur bridges between cysteine residues.