The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins
Graphical abstract
Sodium chloride and ATP dissociate the (horse cyt c)–cardiolipin complex but have no influence on that formed by yeast cyt c. Thus, the complex stability strictly depends on the type of protein utilized. This factor may affect the pro-apoptotic activity of the protein.
Introduction
Cytochrome c (cyt c) is a single-chain hemoprotein acting as electron carrier in mitochondria. It is composed of 104 amino acids (12.4 kDa) and the prosthetic group is heme. In the protein the heme is covalently bound to the polypeptide chain by two thioether bridges formed with residues C14 and C17, while H18 and M80 are the two axial ligands of the heme iron. In the cellular environment the protein functions between the inner and the outer membrane of the mitochondrion, mediating electron transfer between different proteins of the respiratory chain. 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]. This explains why considerable effort has been made over recent years to clarify the mechanisms governing the cyt c–CL interaction and the (cyt c–CL) complex stability.
Two models have been proposed to describe the process leading to (cyt c–CL) complex formation. Both consider that, upon binding, one acyl chain of CL protrudes in the protein interior. 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]. This hypothesis is supported by the fact that CL is the only phospholipid of the mitochondrial membrane which binds the protein firmly [12], [13].
In this paper we investigate the interaction of CL liposomes with ferric cyt c obtained from two distinct sources, horse heart and yeast. 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. Although the horse cyt c–CL interaction is strongly influenced by the two effectors, no effect is observed for the binding reaction of CL liposomes with yeast cyt c. In particular, the fact that sodium chloride dissociates the (horse cyt c)–CL complex but exerts no influence on the (yeast cyt c)–CL complex, suggests that the complex behavior strictly depends on the type of protein utilized. The different behavior shown by the two cytochromes is explained on the basis of the different local structure of the regions involved in the interactions with either CL or ATP.
Section snippets
Materials
Horse heart cyt c (type VI, oxidized form) and cardiolipin, as sodium salt from bovine heart (approx 98% purity, lyophilized powder), were obtained from Sigma Chemical Co (St. Louis, MO, USA) and used without further purification. All reagents were of analytical grade.
Liposome preparation
Aqueous dispersions of CL liposomes were prepared according to a procedure described previously [20]. Briefly, a film of lipid was prepared on the inside wall of a round bottom flask by evaporation of a chloroform solution
Ferric cyt c
The UV–vis spectra of native horse heart and yeast cyt c (Fig. 1, Panel A, traces a) display the characteristic Soret band at 409 nm, Q1 band at 530 nm and a weak charge-transfer (CT) band at 695 nm, indicative of the Met-His axial heme coordination. The UV–vis spectra of the native proteins do not change in the presence of 0.5 M NaCl (Fig. 1, Panel A, traces b) as also confirmed by the RR spectra. The high frequency region spectra of horse heart and yeast cyt c are identical to those obtained in
Discussion
In mitochondrion, a small portion (about 15%) of cyt c is tightly bound to the membrane while the remainder is free or loosely bound [1], [2]. The unbound cyt c participates in eT and actively prevents oxidative stress, while membrane-bound cyt c is mainly involved in cell death. To initiate the apoptotic process the protein acquires peroxidase activity, an event which favors protein dissociation from the mitochondrial membrane [9], [39], [40] and the subsequent release of cyt c into the
List of abbreviations
- CCD
Charge-Coupled Device
- CD
circular dichroism
- CL
cardiolipin
- cmc
critical micelle concentration
- CT
charge transfer
- cyt c
cytochrome c
- IPTG
isopropyl-ß-d-thiogalactopyranoside
- RR
resonance Raman
- UV–vis
Ultraviolet–visible
- wt
wild type
Acknowledgment
This work was supported by local Italian grants (ex 60%) to G.S. and R.S.
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