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.

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Summary Amino acid oxidation, resulting from oxidative stress, is related to certain diseases and aging. To correlate reactive oxygen species with oxidized products of individual amino acids, we oxidized Met, Trp, His, and Tyr by treatment with singlet oxygen, superoxide, or hydroxyl radicals. The structures of oxidized amino acids were determined by nuclear magnetic resonance spectroscopy, infra-red spectroscopy, and fast atom bombardment-mass spectrometry. Among the compounds identified, N-formylkynurenine (NFK), a well-known oxidation product of Trp in vivo via enzymatic and non-enzymatic reactions, and its hydroxylated form were found to be major products of the reaction with singlet oxygen. Since NFK is a pivotal oxidation product of Trp, we raised antiserum against NFK by immunizing a rabbit with NFK-conjugated bovine serum albumin. The resulting antiserum was then used to detect photooxidized trypsin and kynurenine-conjuated ovalbumin as well as NFK-albumin. An antiserum such as this would be useful tool for detecting NFK and kynurenine in situ.