Succinylation of cytochrome c investigated by electrospray ionization mass spectrometry: Reactive lysine residues

The horse heart cytochrome c contains nineteen lysine residues that constitute 18% of the amino acid residues in the protein. These lysine residues are widely spread throughout the structure of the protein. Acylation of cytochrome c with succinic anhydride produces different partially succinylated cytochrome c species due to acylation of various lysine residues present in the protein. Partially succinylated species present in the reaction solutions were detected by electrospray ionization mass spectrometry (ESI-MS). ESI-MS shows that a maximum of seven lysine residue are readily modified on addition of excess succinic anhydride to the protein. Lysine residues that are readily succinylated are identified by ESI-MS detection of the peptide fragments produced by the trypsin digestion of partially succinylated cytochrome c solutions, and by peptide mass mapping. The lysine residues, viz. K86, K79, K72, K60, K39, K27 and K25 were identified as the most reactive residues. These lysine residues are in the locations in the protein that form turn or loop structures. The present results show that the lysines in the helical regions are much less reactive than those in the flexible loops or in the turn regions of the protein structure. It is proposed that the lower reactivity of the lysine residues present in the helical regions may be due to the higher rigidity of the helical regions.