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

doi:10.1016/j.jinorgbio.2011.07.022

Journal of Inorganic Biochemistry

Volume 105, Issue 11, November 2011, Pages 1365-1372

https://doi.org/10.1016/j.jinorgbio.2011.07.022 Get rights and content

Abstract

In cells a portion of cytochrome c (cyt c) (15–20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c–CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)–CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.

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

circular dichroism

cardiolipin

cmc

critical micelle concentration

charge transfer

cyt c

cytochrome c

IPTG

isopropyl-ß-d-thiogalactopyranoside

resonance Raman

UV–vis

Ultraviolet–visible

wild type

Acknowledgment

This work was supported by local Italian grants (ex 60%) to G.S. and R.S.

References (43)

M. Schlame et al.
Prog. Lipid Res.
(2000)
V.E. Kagan et al.
Free Radic. Biol. Med.
(2004)
M. Rytömaa et al.
J. Biol. Chem.
(1995)
Z.T. Schug et al.
Biochim. Biophys. Acta
(2009)
V.E. Kagan et al.
Free Radic. Biol. Med.
(2009)
M. Rytömaa et al.
J. Biol. Chem.
(1994)
M. Rytömaa et al.
J. Biol. Chem.
(1992)
G.W. Bushnell et al.
J. Mol. Biol.
(1990)
G.V. Louie et al.
J. Mol. Biol.
(1990)
U. Subuddhi et al.
Colloids Surf. B Biointerfaces
(2007)

R. Santucci et al.

J. Inorg. Biochem.

(1997)

A.M. Berghuis et al.

J. Mol. Biol.

(1992)

E.K. Tuominen et al.

J. Biol. Chem.

(2001)

H. Bayir et al.

Biochim. Biophys. Acta

(2006)

R.N. Lewis et al.

Biochim. Biophys. Acta

(2009)

E. Kalanxhi et al.

Biochem. J.

(2007)

F. Sinibaldi et al.

Biochemistry

(2008)

F. Sinibaldi et al.

J. Biol. Inorg. Chem.

(2010)

V.E. Kagan et al.

Nat. Chem. Biol.

(2005)

P. Caroppi et al.

Curr. Med. Chem.

(2009)

M. Ott et al.

Cell Death Diff.

(2007)

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SS-20 (Phe-D-Arg-Phe-Lys-NH₂) was used to demonstrate the role of Met⁸⁰-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy.
Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met⁸⁰-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20.
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Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c.
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Interactions of cytochrome c (cyt c) with cardiolipin (CL) play a critical role in early stages of apoptosis. Upon binding to CL, cyt c undergoes changes in secondary and tertiary structure that lead to a dramatic increase in its peroxidase activity. Insertion of the protein into membranes, insertion of CL acyl chains into the protein interior, and extensive unfolding of cyt c after adsorption to the membrane have been proposed as possible modes for interaction of cyt c with CL. Dissociation of Met80 is accompanied by opening of the heme crevice and binding of another heme ligand. Fluorescence studies have revealed conformational heterogeneity of the lipid-bound protein ensemble with distinct polypeptide conformations that vary in the degree of protein unfolding. We correlate these recent findings to other biophysical observations and rationalize the role of experimental conditions in defining conformational properties and peroxidase activity of the cyt c ensemble. Latest time-resolved studies propose the trigger and the sequence of cardiolipin-induced structural transitions of cyt c.

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

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Liposome preparation

Ferric cyt c

Discussion

List of abbreviations

Acknowledgment

Prog. Lipid Res.

Free Radic. Biol. Med.

J. Biol. Chem.

Biochim. Biophys. Acta

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Colloids Surf. B Biointerfaces

J. Inorg. Biochem.

J. Mol. Biol.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochem. J.

Biochemistry

J. Biol. Inorg. Chem.

Nat. Chem. Biol.

Curr. Med. Chem.

Cell Death Diff.