Abstract
The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.
Highlights
Because NO interacts with these radical intermediates, we further studied whether NO acted as an inhibitor of peroxidase activity of cyt c1⁄7CL complexes
We found that 1 h of incubation of cyt c1⁄7TLCL in the presence of H2O2 resulted in accumulation of (165 Ϯ 25) pmol CLOOH/nmol TLCL
Note that multiple oxygenated species of CL, including those containing mono, di, and trihydroperoxides of CL and its hydroxy-derivatives appear after the incubation with cyt c ϩ H2O2
Summary
Interested in the NO regulation of peroxidase activity of cyt c1⁄7CL complex because of its role in induction of apoptosis. NO can act as a regulator of the H2O2-dependent peroxidase activity of the cyt c1⁄7CL complex. Spontaneously released NP ϭ 2 mol of NO/mol of donor molecule with a t1⁄2 of 15 min at 37 °C. 1 mol of HNO was released per mol of Angeli’s salt with a t1⁄2 of 17 min. NO concentration in the solution was estimated from the concentration of NO donor, assuming that NO oxidizes at a normal oxygen concentration ([O2] ϭ 220 M) via bimolecular process with a rate constant kNOox ϭ 103 (MϪ1 sϪ1) [24, 27]. The corresponding equation is as follows, ͱ [NO] ϭ FluxNO/kNOox (Eq 2)
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