Marked difference in cytochrome c oxidation mediated by HO(*) and/or O(2)(*-) free radicals in vitro.
Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N(2)O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK+16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.
Département de Radiothérapie, Centre Antoine Lacassagne, Nice Cedex 2, France.
Juliette
J
Thariat
Fabrice
F
Collin
Catherine
C
Marchetti
Nazha Sid
NS
Ahmed-Adrar
Heidi
H
Vitrac
Daniel
D
Jore
Monique
M
Gardes-Albert
D000595:Amino Acid Sequence; D000818:Animals; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D006736:Horses; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D018384:Oxidative Stress / Q000528:radiation effects; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013481:Superoxides / Q000737:chemistry* / Q000494:pharmacology
D010455:Peptides; D013481:Superoxides; D017665:Hydroxyl Radical; D045304:Cytochromes c