Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis

The reaction of radiolytically generated OH with cytochrome c was investigated by mass spectrometry. Tryptic digestion and characterization of the oxidized peptides by MALDI-TOF and ESI tandem mass spectrometry identified eight different amino acid residues with oxidized side chains with no cleavage of the protein detected. Solvent-accessible aromatic and methionine residues are the most susceptible to oxidation by OH. These results support the careful use of OH in characterizing protein surfaces. Dose–response studies identified the residues most prone to oxidation to be Phe-36, Phe-46, and Met-80. Hydroxylation of Phe-36 and Phe-46 should serve as indicators of the presence of OH in the mitochondrial intermembrane space. Using solutions containing 50 at.% 18O, our study also provides a novel method of determining the source of oxygen during OH-mediated oxidation of proteins and contributes to identification of the modified residue type, with Phe>Tyr>Met in 18O incorporation. During aerobic radiolysis, UV–vis spectroscopy indicates that ferrocytochrome c reaches a steady state concomitant with reduction of the heme.