Probing the Stoichiometry and Oxidation States of Metal Centers in Iron−Sulfur Proteins Using Electrospray FTICR Mass Spectrometry

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Abstract
Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron−sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal−sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak 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]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.

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