Evidence for the Role of a Peroxidase Compound I-type Intermediate in the Oxidation of Glutathione, NADH, Ascorbate, and Dichlorofluorescin by Cytochrome c/H2O2

Abstract

The release of cytochrome c from mitochondria is a crucial step in apoptosis, resulting in the activation of the caspase proteases. A further consequence of cytochrome c release is the enhanced mitochondrial production of superoxide radicals (O 2 . ), which are converted to hydrogen peroxide by manganese-superoxide dismutase. Recently, we showed that cytochrome c is a potent catalyst of 2,7-dichlorofluorescin oxidation to the fluorescent 2,7-dichlorofluorescein by these species, leading to the conclusion that 2,7-dichlorofluorescein fluorescence is a reflection of cytosolic cytochrome c concentration rather than "reactive oxygen species" levels (Burkitt, M. J., and Wardman, P.

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

“…Under these conditions HE was totally consumed within 1 h, despite the fact that the concentration of HE was 10 times higher than that of cyt c 3+ . This was attributed to the peroxidase-like activity of cyt c 3+ [19]. Peaks corresponding to products B and C in Figure 3C are proposed as reaction intermediates and A and E + are designated as final products.…”

“…Oxidant-induced iron signaling was shown to be responsible for oxidation of dichlorodihydrofluorescein (DCFH) to a green fluorescent dichlorofluorescein (DCF) product [23]. Mitochondrial cyt c release was reported to catalyze the oxidation of DCFH to DCF [19,23]. Recently, HE-derived red fluorescence was used to monitor the iron-dependent intracellular oxidative events in neuronal cells [24].…”

Redox Regulation of Tumor Necrosis Factor Signaling

“…Third, antioxidants, especially exogenously added antioxidants, greatly affect DCF fluorescence independent of H 2 O 2 levels (153). Most problematic in the use of DCFH for TNF studies is that cytochrome c can act as a peroxidase and oxidize DCFH to DCF, independent of changes to H 2 O 2 levels (18,84). Because cytochrome c is released during TNFinduced apoptosis (when DCF fluorescence is the greatest), DCF oxidation observed after TNF treatment may be a consequence of cytochrome c release that preceded apoptosis rather than changes in H 2 O 2 levels.…”

Mutant Cu/Zn-Superoxide Dismutase Induced Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis

“…The evidence about deleterious role of increased SOD1 expression has been most recently complemented by studies demonstrating that overexpression o f S O D 1 i n r e t i n a l e a d s t o i n c r e a s e d hydroperoxide levels and accelerated damage of cone cells (Usui et al, n.d.). The key component for the SOD1-derived hydroperoxide toxicity in IMS is cytochrome c. Previous studies, including electron paramagnetic resonance (EPR) studies (Barr et al, 1996;Svistunenko, 2005;Belikova et al, 2006;Basova et al, 2007) have demonstrated that the reaction of cytochrome c with hydroperoxide results in formation of oxoferryl cytochrome c (peroxidase compound I-type intermediate) and corresponding protein-derived tyrosyl radical, which is highly reactive and has a potential to oxidize proteins, DNA, and lipids, as well as endogenous antioxidants such as glutathione, NADH, and ascorbate (Lawrence et al, 2003) (Fig. 3).…”

“…Upon this reaction, hydroperoxide oxidizes the prosthetic heme in the cytochrome c molecule to oxoferryl heme, forming so-called peroxidase compound I-type intermediate, a highly reactive oxidant that is able to react with a number of intracellular targets including proteins, nucleic acids and lipids, causing cell damage (Fig. 3) (Lawrence et al, 2003). Cytochrome c peroxidase activity is controlled by the coordination state of heme iron, particularly by the sulphur ligand of methionine-80 (Met-80), which can be easily displaced by hydroperoxide (Barr et al, 1996;Qian et al, 2002).…”