Insights into Cytochrome c−Cardiolipin Interaction. Role Played by Ionic Strength

Abstract

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.

The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins

“…Previous studies on horse ferricyt c have shown that formation of the (CL-cyt c) complex is coupled with tertiary structural changes in the protein and a marked alteration of the heme pocket [3,5,6,11]. In this paper, the investigation is extended to yeast iso-1-ferricyt c. Fig.…”

“…1 μM [5], as determined by the spectroscopic method based on the fluorescence of 1,6-diphenylhexatriene [23]) was analyzed by following the changes induced in the Soret CD region (400-450 nm) of the protein by stepwise addition of a few microliters of 2.5 mM CL solution to a 10 μM cyt c solution (V i = 1.0 mL; 0.5 cm pathlength cell). The buffer was 25 mM Hepes containing 0.1 mM EDTA, pH 7.0.…”

“…In healthy cells a portion of cyt c (15-20%) remains tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane [1,2]. CL-bound cyt c shows tertiary structural rearrangements, which include alteration of the heme pocket region and detachment of M80 from the sixth coordination position of the heme iron [3][4][5][6]. The CL-specific peroxidase action acquired by membrane-bound cyt c in the early stages of apoptosis, which initiates the protein pro-apoptotic activity, is critical for cells; CL peroxidation induces cyt c release into the cytosol and favors the accumulation of products releasing pro-apoptotic factors [7][8][9][10].…”

“…However, one model identifies the hosting region in the hydrophobic channel close to the invariant residue N52 [3], whereas the other in the M80-containing loop [4]. The fact that the CL liposomes bind cyt c at two distinct binding sites of the protein [5,11] led to the proposal that two acyl chains of CL (instead of one, as suggested by the two above mentioned models) may protrude into cyt c [11]. This event is sterically permitted in view of the unique structure of CL which, contrary to the other phospholipids constituting the mitochondrial membrane, possesses four (instead of two) acyl chains [6].…”

“…The two proteins have a similar tertiary architecture [14][15][16][17], but yeast cyt c displays a significantly lower stability than the protein from horse, it does not bind ATP and, contrary to horse cyt c, lacks pro-apoptotic activity [18,19]. The CL-cyt c binding reaction has been investigated in the absence and in the presence of ATP and sodium chloride, two compounds that significantly influence the (horse cyt c)-CL binding process [5]. The data clearly show that the two proteins behave differently.…”