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

Vilmante Borutaite,Guy C Brown

doi:10.1074/jbc.m700322200

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.

Highlights

Wang [6] that cytochrome c release from mitochondria was central to apoptosis, there has been some question as to whether the two redox states of cytochrome c are effective in promoting caspase activation [7,8,9,10]
We find that mitochondria from apoptotic cells are capable of fully oxidizing cytosolic cytochrome c via cytochrome oxidase
Prior to addition to cytosol, was in the oxidized form, but it was rapidly reduced when added to the cytosolic extracts, coming to a steady state of about 80% reduction after 15 min of incubation

Summary

Introduction

Prior to addition to cytosol, was in the oxidized form, but it was rapidly reduced when added to the cytosolic extracts, coming to a steady state of about 80% reduction after 15 min of incubation. Cytochrome c was preincubated with 120 nM COX, 1.9 ␮M yeast LDH plus 1 mM lactate, or 25 ␮M TMPD for 5 min (where indicated) and 100 ␮g of cytosolic extract was added.

Results

Conclusion