The finding that cytochrome c (cyt c) plays a role in programmed cell death after its release from the mitochondrion has recently renewed interest in this protein. The structural changes in cytochrome c observed at early stages of the apoptotic process have been related to changes occurring in the protein when it forms a complex with phospholipid vesicles. Among the lipids constituting the membrane, cardiolipin is the one thought to bind to cyt c. In this paper, we have investigated the influence exerted by ionic strength on cytochrome c-cardiolipin interaction and found that formation of the cytochrome c-cardiolipin complex occurs via two distinct transitions, implying a high-affinity site and a low-affinity site. Ionic strength significantly influences complex stability; sodium chloride dissociates the complex through two distinct transitions, the second of which occurs at a very high anion concentration. ATP also dissociates the complex, but under the conditions that were investigated, its action is limited to the high-affinity site. The dissociation process is characterized by a very slow kinetic rate constant ( k obs = 4.2 x 10 (-3) s (-1)) and requires several minutes to be completed. We ascribe it to the high activation barrier met by the protein when restoring the native Fe(III)-M80 axial bond. The peroxidase activity shown by cardiolipin-bound cytochrome c is indicative of a less packed protein tertiary conformation in the complex. In line with earlier reports, these data highlight the manifold functions of cytochrome c besides the well-known role it plays in oxidative phosphorylation, shedding more light on the properties of the cytochrome c-cardiolipin complex, involved in the progression of early stages of apoptosis.

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c-CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c-CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c-CL complex.

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c−cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c−cardiolipin interaction, as these side chains appear to be critical for cytochrome c−cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet−visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80−Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c−cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

Apoptosis is strictly connected to the pathogenesis of many human diseases, including neoplastic, neurodegenerative or cardiovascular diseases. It is a highly programmed cell death which can be activated by various factors. Mitochondria play a key role in the apoptotic process; their damage, which involves permeabilization of the outer mitochondrial membrane, activates a series of events that lead to cell death. Of the two proposed signaling pathways of apoptosis, i.e. the 'extrinsic' and the 'intrinsic' pathway, the latter is assumed to initiate in mitochondria. Its activation involves release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space. In the cytosol, cytochrome c exerts its pro-apoptotic action. It binds to the apoptosis protease activation factor (APAf-1) and forms a complex indicated as 'apoptosome'. The complex-induced activation of pro-caspase 9 initiates an enzymatic reaction cascade leading to the execution of apoptosis in cells. This review provides an overview of the key role played by mitochondria and cytochrome c in the activation of the apoptotic process.

No abstract