title: Self-oxidation of cytochrome c at methionine80 with molecular oxygen induced by cleavage of the Met-heme iron bond.
abstract: Met80 of cytochrome c (cyt c) has been shown to dissociate from its heme iron when cyt c interacts with cardiolipin (CL), which triggers the release of cyt c into the cytosol initiating apoptosis. We found that the mass of human cyt c increases by 16 Da in the Met80-Lys86 region by reaction with molecular oxygen in the presence of CL-containing liposomes and dithiothreitol (DTT). To investigate the effect of Met80 dissociation on the reaction of cyt c with molecular oxygen without affecting its secondary structures, a human cyt c mutant (Δ8384 cyt c) was constructed by removing two amino acids (Val83 and Gly84) from the loop containing Met80. According to MALDI-TOF-MS and tandem mass measurements, Met80 of Δ8384 cyt c was modified site-specifically to methionine sulfoxide when purified in the presence of molecular oxygen, whereas Met80 was not modified in the absence of molecular oxygen. A red-shift of the Soret band from 406 to 412 nm and absorption increase at ∼536 and ∼568 nm were observed for Δ8384 cyt c when it reacted with DTT and molecular oxygen, followed by a further red-shift of the Soret band to 416 nm and absorption increase at ∼620 and ∼650 nm. These results indicate that Met80 of cyt c is oxidized site-specifically by formation of the oxy and subsequent compound I-like species when Met80 dissociates from the heme iron, where the Met80 modification may affect its peroxidase activity related to apoptosis.
authors.affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. hirota@ms.naist.jp.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Wang
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ando
authors.forename: Ari Dwi
authors.initials: AD
authors.lastname: Nugraheni
authors.forename: Chunguang
authors.initials: C
authors.lastname: Ren
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagao
authors.forename: Shun
authors.initials: S
authors.lastname: Hirota
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008715:Methionine / Q000031:analogs & derivatives / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D008081:Liposomes; D006418:Heme; D045304:Cytochromes c; D008715:Methionine; D007501:Iron; D010100:Oxygen; D004229:Dithiothreitol; C013111:methionine sulfoxide