Mitochondrial Regulation of Caspase Activation by Cytochrome Oxidase and Tetramethylphenylenediamine via Cytosolic Cytochrome c Redox State

Abstract

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.

Tetramethylphenylenediamine protects the isolated heart against ischaemia‐induced apoptosis and reperfusion‐induced necrosis

“…We have previously shown that TMPD and related agents can block caspase activation in cultured cells and cytosolic extracts by reducing the cytochrome c (from c 3+ to c 2+ ), which is then less able to activate the apoptosome (Brown and Borutaite, 2008). TMPD did not significantly inhibit caspases themselves after activation (Borutaite and Brown, 2007), and did not inhibit measured …”

“…Cytochrome c release activates apoptosis by binding to a cytosolic adaptor protein, APAF-1, which then recruits and activates procaspase-9 within the apoptosome (Li et al, 1997). Recently, we and others have shown that the reduced form of cytochrome c (c 2+ ) has little or no ability to activate the caspases, and that factors that keep the cytosolic cytochrome c reduced during apoptosis induction prevent caspase activation and death of cultured cell lines (Pan et al, 1999;Suto et al, 2005;Borutaite and Brown, 2007). One such factor is N,N,N′,N′-tetramethylphenylene-1,4-diamine (TMPD), which readily crosses cell membranes and rapidly reduces cytochrome c, and can be re-reduced by a number of cellular reductases or reductants (Sarti et al, 1992).…”

Downregulation of cytochrome c oxidase 1 induced radioresistance in esophageal squamous cell carcinoma

“…The redox state of cytochrome c is important in this caspase-dependent apoptosis. Only oxidized cytochrome c can activate the apoptosome, whereas reduced cytochrome c cannot (11)(12)(13). The most potent enzyme that oxidizes cytochrome c is cytochrome c oxidase (13).…”

“…Only oxidized cytochrome c can activate the apoptosome, whereas reduced cytochrome c cannot (11)(12)(13). The most potent enzyme that oxidizes cytochrome c is cytochrome c oxidase (13). Multiple caspase-dependent and -independent cell death pathways induce apoptosis after DNA damage (14).…”

A role for human neuroglobin in apoptosis

“…2a). In the activation phase, it has been demonstrated that only ferric cytochrome c is active in the assembly of the apoptosome (50)(51)(52)(53). Based on this observation and the redox reactions of neuroglobin described above, it was hypothesised that neuroglobin might prevent cell death by intervention in the apoptotic pathway by reducing released mitochondrial cytochrome c to the inactive ferrous form (54).…”