Cytochrome c and glutathione (GSH) are two important biomolecules that regulate many cellular processes. The reaction of cytochrome c with GSH involves radical oxygen species and exhibits significant complexity. In the present work, the reaction of cytochrome c with GSH in water was characterized using mass spectrometry. The results show for the first time that the reaction generates Multiple products including apocytochrome c in oxidized and reduced forms, glutathionylated apocytochroine c, GSH-modified cytochrome c, and oxidized and hydroxylated species. The reaction is O-2 dependent and is rapid in water at neutral pH and 37 C. The reaction involves the cleavage of thioether linkages between the heme and apocytochrome c. Evidence for the role of H2O2 and other oxygen radicals in this reaction is also provided. (c) 2006 Elsevier Inc. All rights reserved.