Suppression of the pro-apoptotic function of cytochrome c by singlet oxygen via a haem redox state-independent mechanism.
Stimuli for apoptotic signalling typically induce release of cyt c (cytochrome c) from mitochondria. Cyt c then initiates the formation of the apoptosome, comprising Apaf-1 (apoptotic protease-activating factor 1), caspase-9 and other cofactors. The issue of whether the redox state of the haem in cyt c affects the initiation of the apoptotic pathway is currently a subject of debate. In a cell-free reconstitution system, we found that only oxidized cyt c was capable of activating the caspase cascade. Oxidized cyt c was reduced by the physiological reductants cysteine and glutathione, after which it was unable to activate the caspase cascade. It is thus likely that cyt c with oxidized haem is in a conformation capable of interaction with Apaf-1 and forming apoptosomes. When either oxidized or reduced cyt c was treated with submillimolar concentrations of endoperoxide, which affected less than 3% of the redox state of haem, the ability of the oxidized cyt c to activate the caspase cascade was abolished. Higher amounts of singlet oxygen were required to affect the optical spectral change of haem, suggesting that the suppressed pro-apoptotic function of oxidized cyt c is a mechanism that is separate from the redox state of haem. Oxidative protein modification of cyt c by singlet oxygen was evident, on the basis of elevated contents of carbonyl compounds. Our data suggest that singlet oxygen eliminates the pro-apoptotic ability of oxidized cyt c not via the reduction of haem, but via the modification of amino acid residues that are required for apoptosome formation.
Suto
Daisuke
D
Department of Biomolecular Function, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan.
Sato
Kazuaki
K
Ohba
Yoshihiro
Y
Yoshimura
Tetsuhiko
T
Fujii
Junichi
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D003545:Cysteine / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D006418:Heme / Q000378:metabolism*; D006736:Horses; D006801:Humans; D010084:Oxidation-Reduction / Q000187:drug effects; D026082:Singlet Oxygen / Q000378:metabolism / Q000494:pharmacology*
D026082:Singlet Oxygen; D006418:Heme; D045304:Cytochromes c; D020169:Caspases; D005978:Glutathione; D003545:Cysteine