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First published online 5 February 2008
doi: 10.1242/jcs.021303

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Evaluating cytochrome c diffusion in the intermembrane spaces of mitochondria during cytochrome c release

Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK

^* Author for correspondence (e-mail: m.crompton{at}biochemistry.ucl.ac.uk)

Accepted 19 November 2007

Truncated Bid (tBid) releases cytochrome c from mitochondria by inducing Bak (and Bax) pore formation in the outer membrane. An important issue is whether a second tBid action, independent of Bak and Bax, is also required to enhance cytochrome c mobility in the intermembrane spaces. To investigate this, we developed a kinetic analysis enabling changes in the diffusibility of cytochrome c in the intermembrane spaces of isolated mitochondria to be differentiated from changes resulting from Bak activation. Cytochrome c diffusibility in the intermembrane spaces was unaffected by changes in [tBid] over the range 0.5-19.0 pmol per mg of mitochondrial protein, when tBid-dependent Bak activation was increased several-thousand fold. However, high [tBid] (100 pmol mg^–1) did increase diffusibility by approximately twofold. This was attributable to the permeability transition. Basal cytochrome c diffusibility in the intermembrane spaces in the absence of tBid was determined to be approximately 0.2 minute^–1, which is sufficient to support cytochrome c release with a half-time of 3.4 minutes. It is concluded that tBid has a monofunctional action at low concentrations and, more generally, that the basal cytochrome c diffusibility in the intermembrane spaces is adequate for rapid and complete cytochrome c release irrespective of the mode of outer membrane permeabilisation.

Key words: Bid, Bak, Cytochrome c, Diffusion, Mitochondria, Apoptosis