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The interactions of ferric and ferrous cytochrome c with cardiolipin in phospholipid membranes studied by solid-state 2H and 31P NMR
Journal of
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 441 (1998) 183-188
The interactions of ferric and ferrous cytochrome c with cardiolipin in phospholipid membranes studied by solid-state 2H and 31p NMR Suhk-mann Kim, Kyong-hwa Shin, Toshimichi Fujiwara, Hideo Akutsu* Department of Bioengineering, Faculty of Engineering, YokohamaNational University. Hodogaya-ku, Yokohama240. Japan
Received 24 February 1997; revised 23 April 1997; accepted 23 April 1997
Abstract
The interactions of ferric and ferrous cytochrome c with cardiolipin (CL) in lipid bilayers were investigated by solid-state 2H and 31p NMR. To examine the effect of the interaction on the glycerol backbone of CL, its glycerol moiety was specifically deuterated. 2H NMR experiment showed that only ferricytochrome c interacts strongly with CL in the CL bilayers and binary mixed phosphatidylcholine(PC)/CL bilayers. This was consistent with the result of cytochrome c binding experiment. Ferricytochrome c binds to CL liposomes as much as two times of ferrocytochrome c. This suggests that the charge of the heme iron is also involved in the interaction. The change of the deuterium quadrupole splitting of CL on binding of cytochrome c was larger for the single component CL bilayer than for the PC/CL bilayer, suggesting that a CL domain rather than a single molecule is responsible for the strong interaction with cytochrome c. © 1998 Elsevier Science B.V. Keywords: Lipid-protein interaction; Cytochrome c; Cardiolipin; 2H NMR; 31p NMR
1. Introduction
Cytochrome c, a highly basic protein ( = 12 kDa) is known as a peripheral membrane protein involved in the electron transport system operating in the inner mitochondrial membrane [1,2]. The lipid-protein interactions would have relevant functional effects on the efficiency o f electron transfer. For instance, cytochrome c interacts strongly with negatively charged phospholipid [3-5]. In particular, its binding to cardiolipin bilayers has been well investigated [6,7]. Cardiolipin is required for the activity o f some membrane proteins such as cytochrome c oxidase [8-10]. It has been also reported that cardiolipin facilitates the binding o f cytochrome c to cytochrome * To whom all correspondenceshould be addressed.
c oxidase [11]. Although cardiolipin constitutes only a small fraction o f the total mitochondrial lipids, it plays a significant role in the electron transport. Therefore, it is important to characterize the specific interactions o f cardiolipin with cytochrome c. Interactions o f cytochrome c, mostly oxidized one, with lipid monolayers and bilayers have been extensively studied [ 12-14]. But the nature of the interactions have not been elucidated yet. Some experiments have been interpreted as showing partial penetration of the cytochrome c into bilayers [ 1,3], whereas others have lead to a contrary conclusion [15,16]. Differences in the mode o f interaction o f oxidized and reduced cytochromes c with phospholipids have been discussed as well. On the basis of measurements o f bilayer thickness, the effect of sodium chloride in releasing the protein from lipids, and the absorption
0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PII S0022-2860(97)00255-X
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spectrum of the protein, Letellier and Shechter have concluded that while ferricytochrome c interacts electrostatically with cardiolipin molecules, the ferrous one interacts hydrophobically to some extent [17]. However, others have reported that ferricytochrome c binds to phosphatidic acid (PA) bilayers through both electrostatic and hydrophobic interactions [18]. A series of NMR studies on the interaction of cytochrome c with the polar head groups of phospholipids have been carried out by Watts and his coworkers [5,19-22]. It was suggested that a specific interaction between cardiolipin and cytochrome c induces reversible protein unfolding. We have examined the interaction by 2H NMR using phospholipids specifically deuterated in the glycerol backbone [23]. The results showed that ferricytochrome c strongly interacts with cardiolipin in the presence of phosphatidylcholine, but the interaction was suppressed by the presence of phosphatidylethanolamine in the lipid bilayers. In this work, we have characterized the interaction of ferri- and ferrocytochrome c with cardiolipin by measuring 2H and 31p NMR spectra. The results showed that there is a difference in the mode of interaction for ferri- and ferrocytochrome c.
2. Materials and m e t h o d s
2.1. Specific deuteration of the glycerol moieties of cardiolipin Synthesis of perdeuterated glycerol ([2Hs]-glycerol) was performed as described elsewhere [24]. The deuterated glycerol was incorporated into the cells of an E. coli mutant requiring glycerol (E. coli K-12 GRA) at 37°C. Phosphatidylethanolamine (PE) and cardiolipin (CL) were extracted from the cells according to the reported method [24]. The extent of deuteration was estimated from a JH NMR spectrum of PE. It was about 70% on average. Phosphatidylcholine (PC) was synthesized by methylation of PE purified from the cells according to the reported method [251.
2.2. Purification of cytochrome c
oxidized form by addition of an excess of K3Fe(CN)6 and then purified by ion-exchange column chromatography on Whatman CM-32, eluted with 0.5 M NaC1, 10 mM phosphate buffer at pH 7.0. Eluent containing the purified protein was concentrated by ultrafiltration using Amicon YM-3 membranes and then dialyzed extensively to remove phosphate [20].
2.3. Preparation of NMR samples Phospholipids were dissolved in chloroform/ methanol (1:1, v/v) and washed with 0.5 vol. of a 0. 5 M Na2SO4, 2.0 mM EDTA solution (pH 7.2) to remove polyvalent metal ions. The chloroform fraction was transferred to a 5 mmq~ tube then dried to a film under nitrogen gas. It was further dried under high vacuum for overnight to remove all traces of organic solvent. Multilamellar liposomes were prepared by dispersing the dry lipid film to 5 mM TrisHCI buffer (pH 7.4) with and without ferri- or ferrocytochrome c in deuterium depleted water (less than 0.2 ppm, CEA) at 50°C. To reduce cytochrome c, a trace of sodium dithionite was added. The reduction was confirmed by the absorption spectrum. Typically, about 40 mg of deuterated lipid was used for 2H NMR measurement. The lipid-protein complexes were obtained by centrifugation at 3000 rpm (Tomy TS-7 rotor) at 4°C. The amount of bound protein was determined from the amount of remaining protein in the supernatant. The absorption spectra were measured with a Shimadzu UV-2000 spectrophotometer. Sample preparation was carried out in a nitrogen atmosphere to prevent the oxidation of lipid fatty acyl chains and of ferrocytochrome c. The molar ratio of cardiolipin/protein in the complexes used in the present study was in the range of 10-35.
2.4. NMR measurements 1H NMR spectra were obtained with a Bruker AM400 spectrometer. 2H NMR experiments were performed with a Chemagnetics CMX-400 spectrometer operating at 61.1 MHz equipped with a CP/MAS probe for a 5 mm4, tube. The 90 ° pulse width was 5/xs. The quadrupole echo pulse sequence (90x Tl-90y--r2) was employed with 7"1 30 gs, r2 = 20 txs and 0.5 s of recycle time. 31p NMR spectra were recorded on a Chemagnetics CMX-400 spectrometer operating =
Prior to use, cytochrome c from horse heart (type VI, Sigma Chemical Co.) was converted to the fully
S. Kim et al./Journal o f Molecular Structure 441 (1998) 183-188
at 161.15MHz under proton decoupling. The decoupling strength was 50kHz. The 90 ° pulse width was 5 t~s for 31p. Chemical shifts were shown in the spectrum relative to 85% phosphoric acid.
3. Results
3.1. Binding of cytochrome c to phospholipid bilayers including cardiolipin The amount of cytochrome c bound to the lipid bilayers is shown in Table 1. Ferricytochrome c binds to cardiolipin (CL) bilayers by as much as two times as much as ferrocytochrome c does. This can be attributed to the loss of one positive formal charge at the heme and/or to a structural change of cytochrome c on reduction. Our previous results showed that ferricytochrome c binds more strongly to phosphatidylcholine(PC)/CL than to phosphatidylethanolamine (PE)/CL bilayers [23]. Since the amount of the bound cytochrome c per cardiolipin molecule is similar for PC/CL and PE/CL bilayers, the net negative charge on a liposome seems to be important for the binding of cytochrome c. Less ferricytochrome c binds to CL liposomes than to PC/CL liposomes. This suggests that the size of the binding site on the lipid bilayer for a single protein molecule is smaller than the area of the lipid bilayer covered by a protein molecule. In CL liposomes, there would be free CL molecules in the area covered by cytochrome c. They are inactive for the binding. Hence, this makes the binding efficiency of the CL liposome lower than that of the PC/CL liposome.
Table 1 The amount of cytochrome c bound to phospholipid liposomes Complexes a
Molar ratio of the bound cytochrome c to cardiolipin
CL-cyt c (ox) CL-cyt c (red) (PE/CL)-cyt c (ox) (PC/CL)-cyt c (ox)
0.067 0.033 0.094 0.093
CL, cardiolipin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; cyt c, cytochrome c; ox, oxidized form; red, reduced form. PC/CL stands for the liposomes of the binary mixture of PC and CL (4:1 w/w).
185
3.2. Deuterium quadrupole splittings of the CL glycerol backbones of the cytochrome c-lipid bilayer complexes The interactions of ferri- and ferrocytochrome c with the deuterated cardiolipin (CL*) in the bilayers were examined by 2H NMR. Fig. I(A) and (D) represent 2H NMR spectra of CL* and PC/CL* bilayers at 40°C. The assignment is given on the top of Fig. I(F) [24,26,27]. For example, 3S stands for the pro-S deuteron at the C-3 site of the glycerol backbone. The 2H NMR spectra in Fig. 1 are the superimposed powder patterns characteristic for nuclei with 1 = 1 under strong magnetic fields. The 2H NMR spectra of CL* and PC/CL* bilayers with ferri- or ferrocytochrome c are also presented in Fig. 1. The quadrupole splittings of the deuterons of CL* in the presence and absence of cytochrome c were plotted in Fig. 2 as a function of temperature. The largest error of the quadrupole splitting was -0.5 kHz for 3S deuterons. Although the changes of the quadrupole splittings are not significant, all quadrupole splittings of the glycerol deuterons of CL became smaller on binding of ferricytochrome c. This is consistent with our previous report [23]. In contrast to it, the quadrupole splittings did not change at all on binding of ferrocytochrome c. The effect of the binding of cytochrome c is similar for the single component and binary mixture bilayers. It was also reported that the quadrupole splittings of the PC component in PC/CL bilayers did not change on binding of ferricytochrome c [23].
3.3. 31p NMR spectra of the cytochrome c-lipid bilayer complexes To monitor the polymorphic structure of the samples and the effect of intermolecular interactions on the polar head groups on binding of cytochrome c, 31p NMR spectra were obtained for the same samples that were used for 2H NMR measurements. The 31p NMR spectra of CL* and PC/CL* bilayers with and without ferri- or ferrocytochrome c are shown in Fig. 3. Their powder patterns indicate that most of phospholipids are in the liquid-crystalline lamellar phase in all samples although there is a small contribution from the isotropic phase (at about 4ppm). The 31p chemical shift anisotropies ( a l l - a±) at 35°C were -31 and
186
S. Kim et al./Journal of Molecular Structure 441 (1998) 183 188
3S 2and3R !
(B)(A)~ 30
'
0 kHz
-30
30
0 kHz
-30
Fig. 1.2H NMR spectra of the deuterated glycerol backbone of cardiolipin(CL) with and without cytochrome c at 61.1 MHz and 40°C: (A) CL bilayers, (B) CL bilayers with ferricytochrome c, (C) CL bilayers with ferrocytochrome c, (D) phosphatidylcholine(PC)/CL (4:1 w/w) bilayers, (E) PC/CL (4:1 w/w) bilayers with ferricytochrome c, (F) PC/CL (4:1 w/w) bilayers with ferrocytochrome c. The assignment is given on the top of the spectrum F. Head denotes the head group of cardiolipin and the other assignments are for the prochiral deuterons of the glycerol backbone.
-43 ppm for CL* and PC/CL* bilayers, respectively. Little change was observed on binding of cytochrome c. The spectra of PC/CL* bilayers clearly consist of two axially symmetric powder patterns with different chemical shift anisotropies. The assignment of these superimposed powder patterns was reported previously [21,23,28,29]. Namely, the powder patterns with large and small chemical shift anisotropies can be ascribed to PC and CL, respectively.
Line broadening of the powder pattern of CL was observed on binding of ferri- or ferrocytochrome c. Especially, the effect of the ferrocytochrome c is significant (Fig. 3(B)). The 31p NMR spectra of the CL bilayers at 30 and 50°C are shown in Fig. 4. The linewidth became broader with an increase of temperature both for ferri- and ferrocytochrome c complexes. This fact suggests that the exchange rate between the bound and free cardiolipins is close either to the 30
-~ 30-
3S =.~_
3S
25-
25-
r.~
~. 20-
E
2 and 3R
15-
Head
2W
2 and 3R
15-
Head
.r-
IC
3'0
5b (A)
lOl
Temperature/°C
3'0
£
5b
(B)
Fig. 2. Temperature dependence of the deuterium quadrupole splittings of the deuterated glycerol backbone of cardiolipin: (A) cardiolipin bilayers, (B) phosphatidylcholine/cardiolipin (4:1 w/w) bilayers. Circles, without cytochrome c; triangles, with ferricytochrome c; crosses, with ferrocytochrome c.
S. Kim et al./Journal of Molecular Structure 441 (1998) 183-188
,
,
i
[
,
25
,
i
,
I
'
i
0
,
,
I
'
'
'
'
-25 ppm
'
'
I
'
'
25
'
'
I
i
,
0
,
,
I
'
187
'
'
-25 ppm
Fig. 3. Proton decoupled 31p NMR spectra of cardiolipin(CL) and phosphatidylcholine(PC)/CLbilayers at 161.15 MHz and 35°C: (A) CL bilayers, (B) CL bilayers with ferrocytochrome c, (C) CL bilayers with ferricytochrome c, (D) PC/CL bilayers, (E) PC/CL bilayers with ferrocytochrome c, (F) PC/CL bilayers with ferricytochrome c. chemical shift difference or to the proton decoupling strength [30]. Since the linewidth is broader for ferrothan for ferricytochrome c complexes at the same temperature, the exchange rate should be faster for the former than the latter in view o f the temperature dependence and mechanisms mentioned above. The exchange rate between the bound and free cardiolipins is much slower than the chemical shift difference or the proton decoupling strength for the ferricytochrome c - P C / C L complex at 35°C.
4. Discussion The present results showed that the only oxidized cytochrome c interacts strongly with CL in the CL
25
0 (A)
-25 ppm
25
0 (B)
bilayers and PC/CL bilayers. It was reported that ferricytochrome c specifically interacts with CL in the PC/CL bilayers but does not with CL in the PE/CL bilayers [23]. The difference was attributed to the molecular miscibility o f the two systems. Namely, while PE and CL are completely miscible, PC and CL are not [31]. In the former mixture, the strong i n t e r - p o l a r - h e a d group interaction among PE and CL, which results in a homogeneous mixing, suppresses the strong interaction between cytochrome c and CL [23]. In the mixture o f PC and CL, the presence o f microdomains was suggested [31]. However, it was not clear at that time that either CL in a PC microdomain or CL in a CL microdomain interacts with cytochrome c. Since the change o f the deuterium quadrupole splittings o f CL* on binding
,,w
-25 ppm
i , , i , , , ,
25
j,,,,
0 (C)
i,,,
-25 ppm
Fig. 4. Proton decoupled 31 P NMR spectra of CL with and without cytochrome c at 30 and 50°C: (A) cardiolipin(CL) bilayers, (B) CL bilayers with ferricytochrome c, (C) CL bilayers with ferrocytochrome c.
188
S. Kim et al./Journal of Molecular Structure 441 (1998) 183-188
of cytochrome c was larger for the single component (CL) bilayers than for the PC/CL bilayers, it can be concluded that CL in the CL microdomain should be responsible for the strong interaction with cytochrome c. In view of earlier reports, the cytochrome c-lipid interaction may affect the domain size as well. It was indicated that the binding of cytochrome c induces segregation of CL in the mixed lipid bilayers [1,32], suggesting that cytochrome c does not associate with the bulk component of the inner mitochondrial membrane, but interacts in a more specific way with cardiolipin. CL also can form a receptor surface in the inner membrane for the mitochondrial creatine kinase enzyme [ 10]. It has been proposed that the interaction of cytochrome e with lipids induce an extended lipid conformation in which the two acyl chains of a lipid point the opposite directions from the head group [33-37]. On the basis of such a model, a hydrophobic interaction between the protein and the inserted hydrocarbon chain can take place at the membrane surface without penetration of the protein into the hydrocarbon region [34,36-38]. Thus, peripheral proteins can be anchored to the membrane. In this conformation, the torsion angle among C2-C3 of the glycerol backbone of the lipid should be in the antiperiplanar range. This kind of conformation can induce a structural distortion around it. This can be one explanation for the strong interaction between ferricytochrome c and cardiolipin. Another possibility is that ferricytochrome c penetrates deeper into the membrane than ferrocytochrome c. Anyhow, the difference in the interaction between cytochrome c and lipid bilayers for the oxidized and reduced forms may play a certain role in the respiratory system in mitochondria. References [1 ] L.R. Brown, K. WiJthrich, Biochim. Biophys. Acta 468 (1977) 389. [2] M. Ryt6maa, P. Mustonen, P.K.J. Kinnunen, J. Biol. Chem. 267 (1992) 22243. [3] A. Rietveld, P. Sijens, A.J. Vekkleij, B. de Kruijff, EMBO J. 12 (1983) 907. [4l M.C. Waltham, B.A. Cornell, R. Smith, Biochim. Biophys. Acta 862 (1986) 451. [5] T.J.T. Pinheiro, A. Watts, Biochemistry 33 (1994) 2451.
[6] B. de Kruijff, P.R. Cullis, Biochirn. Biophys. Acta 602 (1980) 477. [7] R.A. Demel, W. Jordi, H. Lambrechts, H. van Damme, R. Hovius, B. de Kruijff, J. Biol. Chem. 264 (1989) 3988. [8] N.C. Robinson, F. Strey, L.H. Talbert, Biochemistry 19 (1980) 3656. [9] M. Miiller, R. Moser, D. Cheneval, E. Carafoli, J. Biol. Chem. 260 (1985) 3839. [10] D. Marsh, G.L. Powell, Bioelectrochem. Bioenerg. 20 (1988) 73. [l 1] S.B. Vik, G. Georgevich, R.A. Capaldi, Proc. Natl. Acad. Sci. USA 78 (1981) 1456. [12] H.K. Kimbelberg, Mol. Cell. Biochem. 10 (1976) 171. [13] J.M. Boggs, Membrane Fludity Biol. 2 (1983) 89. [14] P.F. Devaux, M. Seigneuret, Biochim. Biophys. Acta 822 (1985) 63. [15] J. Sedzik, A.E. Blaurock, M. H~chli, J. Mol. Biol. 174 (1984) 385. [16] W. MacNaughtan, K.A. Snook, E. Caspi, N.P. Franks, Biochim. Biophys. Acta 818 (1985) 132. [17] L. Letellier, E. Shechter, Eur. J. Biochem. 40 (1973) 507. [18] L. Mateu, F. Caron, V. Luzzati, A. Billecocq, Biochim. Biophys. Acta 508 (1978) 109. [19] T.J.T. Pinheiro, A. Watts, Biochemistry 33 (1994) 2459. [20] P.J.R. Spooner, A. Watts, Biochemistry 30 (1991) 3871. [21] P.J.R. Spooner, A. Watts, Biochemistry 31 (1992) 10129. [22] P.J.R. Spooner, A. Watts, Biochemistry 30 (1991) 3880. [23] K. Shin, T. Fujiwara, H. Akutsu, J. Mol. Struct. 355 (1995) 47. [24] W. Yoshikawa, H. Akutsu, Y. Kyogoku, Y. Akamatsu, Biochim. Biophys. Acta 944 (1988) 321. [25] H. Akutsu, Y. Suezaki, W. Yoshikawa, Y. Kyogoku, Biochim. Biophys. Acta 854 (1986) 213. [26] L.M. Strenk, P.W. Westerman, J.W. Doane, Biophys. J. 48 (1985) 765. [27] P.R. Allegrini, G. Pluschke, J. Seelig, Biochemistry 23 (1984) 6452. [28] R. Ghosh, Biochemistry 27 (1988) 7750. [29] F.M. Marassi, P.M. Macdonald, Biochemistry 30 (1991) 10558. [30] W.P. Rothwell, J.S. Waugh, J. Chem. Phys. 74 (5) (1981) 2721. [31] K. Shin, H. Maeda, T. Fujiwara, H. Akutsu, Biochim. Biophys. Acta 1238 (1995) 42. [32] G.B. Birrell, O.H. Griffith, Biochemistry 15 (1976) 2925. [33] P.K.J. Kinnunen, A. K~iv, J.Y.A. Lehtonen, M. Ryt6maa, P. Mustonen, Chem. Phys. Lipids 73 (1994) 181. [34] P.K.J. Kinnunen, Chem. Phys. Lipids 63 (1992) 251. [35] H. Hauser, 1. Pascher, S. Sundell, Biochemistry 27 (1988) 9166. [36] M. Hong, K. Schmidt-Rohr, H. Zimmermann, Biochemistry 35 (1996) 8335. [37] E. Kahana, J.C. Pinder, K.S. Smith, W.B. Gratzer, Biochem. J. 282 (1992) 75. [38] M. Ryt6maa, P.K.J. Kinnunen, J. Biol. Chem. 270 (1995) 3197.
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 441 (1998) 183-188
The interactions of ferric and ferrous cytochrome c with cardiolipin in phospholipid membranes studied by solid-state 2H and 31p NMR Suhk-mann Kim, Kyong-hwa Shin, Toshimichi Fujiwara, Hideo Akutsu* Department of Bioengineering, Faculty of Engineering, YokohamaNational University. Hodogaya-ku, Yokohama240. Japan
Received 24 February 1997; revised 23 April 1997; accepted 23 April 1997
Abstract
The interactions of ferric and ferrous cytochrome c with cardiolipin (CL) in lipid bilayers were investigated by solid-state 2H and 31p NMR. To examine the effect of the interaction on the glycerol backbone of CL, its glycerol moiety was specifically deuterated. 2H NMR experiment showed that only ferricytochrome c interacts strongly with CL in the CL bilayers and binary mixed phosphatidylcholine(PC)/CL bilayers. This was consistent with the result of cytochrome c binding experiment. Ferricytochrome c binds to CL liposomes as much as two times of ferrocytochrome c. This suggests that the charge of the heme iron is also involved in the interaction. The change of the deuterium quadrupole splitting of CL on binding of cytochrome c was larger for the single component CL bilayer than for the PC/CL bilayer, suggesting that a CL domain rather than a single molecule is responsible for the strong interaction with cytochrome c. © 1998 Elsevier Science B.V. Keywords: Lipid-protein interaction; Cytochrome c; Cardiolipin; 2H NMR; 31p NMR
1. Introduction
Cytochrome c, a highly basic protein ( = 12 kDa) is known as a peripheral membrane protein involved in the electron transport system operating in the inner mitochondrial membrane [1,2]. The lipid-protein interactions would have relevant functional effects on the efficiency o f electron transfer. For instance, cytochrome c interacts strongly with negatively charged phospholipid [3-5]. In particular, its binding to cardiolipin bilayers has been well investigated [6,7]. Cardiolipin is required for the activity o f some membrane proteins such as cytochrome c oxidase [8-10]. It has been also reported that cardiolipin facilitates the binding o f cytochrome c to cytochrome * To whom all correspondenceshould be addressed.
c oxidase [11]. Although cardiolipin constitutes only a small fraction o f the total mitochondrial lipids, it plays a significant role in the electron transport. Therefore, it is important to characterize the specific interactions o f cardiolipin with cytochrome c. Interactions o f cytochrome c, mostly oxidized one, with lipid monolayers and bilayers have been extensively studied [ 12-14]. But the nature of the interactions have not been elucidated yet. Some experiments have been interpreted as showing partial penetration of the cytochrome c into bilayers [ 1,3], whereas others have lead to a contrary conclusion [15,16]. Differences in the mode o f interaction o f oxidized and reduced cytochromes c with phospholipids have been discussed as well. On the basis of measurements o f bilayer thickness, the effect of sodium chloride in releasing the protein from lipids, and the absorption
0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PII S0022-2860(97)00255-X
184
S. Kim et al./Journal of Molecular Structure 441 (1998) 183 188
spectrum of the protein, Letellier and Shechter have concluded that while ferricytochrome c interacts electrostatically with cardiolipin molecules, the ferrous one interacts hydrophobically to some extent [17]. However, others have reported that ferricytochrome c binds to phosphatidic acid (PA) bilayers through both electrostatic and hydrophobic interactions [18]. A series of NMR studies on the interaction of cytochrome c with the polar head groups of phospholipids have been carried out by Watts and his coworkers [5,19-22]. It was suggested that a specific interaction between cardiolipin and cytochrome c induces reversible protein unfolding. We have examined the interaction by 2H NMR using phospholipids specifically deuterated in the glycerol backbone [23]. The results showed that ferricytochrome c strongly interacts with cardiolipin in the presence of phosphatidylcholine, but the interaction was suppressed by the presence of phosphatidylethanolamine in the lipid bilayers. In this work, we have characterized the interaction of ferri- and ferrocytochrome c with cardiolipin by measuring 2H and 31p NMR spectra. The results showed that there is a difference in the mode of interaction for ferri- and ferrocytochrome c.
2. Materials and m e t h o d s
2.1. Specific deuteration of the glycerol moieties of cardiolipin Synthesis of perdeuterated glycerol ([2Hs]-glycerol) was performed as described elsewhere [24]. The deuterated glycerol was incorporated into the cells of an E. coli mutant requiring glycerol (E. coli K-12 GRA) at 37°C. Phosphatidylethanolamine (PE) and cardiolipin (CL) were extracted from the cells according to the reported method [24]. The extent of deuteration was estimated from a JH NMR spectrum of PE. It was about 70% on average. Phosphatidylcholine (PC) was synthesized by methylation of PE purified from the cells according to the reported method [251.
2.2. Purification of cytochrome c
oxidized form by addition of an excess of K3Fe(CN)6 and then purified by ion-exchange column chromatography on Whatman CM-32, eluted with 0.5 M NaC1, 10 mM phosphate buffer at pH 7.0. Eluent containing the purified protein was concentrated by ultrafiltration using Amicon YM-3 membranes and then dialyzed extensively to remove phosphate [20].
2.3. Preparation of NMR samples Phospholipids were dissolved in chloroform/ methanol (1:1, v/v) and washed with 0.5 vol. of a 0. 5 M Na2SO4, 2.0 mM EDTA solution (pH 7.2) to remove polyvalent metal ions. The chloroform fraction was transferred to a 5 mmq~ tube then dried to a film under nitrogen gas. It was further dried under high vacuum for overnight to remove all traces of organic solvent. Multilamellar liposomes were prepared by dispersing the dry lipid film to 5 mM TrisHCI buffer (pH 7.4) with and without ferri- or ferrocytochrome c in deuterium depleted water (less than 0.2 ppm, CEA) at 50°C. To reduce cytochrome c, a trace of sodium dithionite was added. The reduction was confirmed by the absorption spectrum. Typically, about 40 mg of deuterated lipid was used for 2H NMR measurement. The lipid-protein complexes were obtained by centrifugation at 3000 rpm (Tomy TS-7 rotor) at 4°C. The amount of bound protein was determined from the amount of remaining protein in the supernatant. The absorption spectra were measured with a Shimadzu UV-2000 spectrophotometer. Sample preparation was carried out in a nitrogen atmosphere to prevent the oxidation of lipid fatty acyl chains and of ferrocytochrome c. The molar ratio of cardiolipin/protein in the complexes used in the present study was in the range of 10-35.
2.4. NMR measurements 1H NMR spectra were obtained with a Bruker AM400 spectrometer. 2H NMR experiments were performed with a Chemagnetics CMX-400 spectrometer operating at 61.1 MHz equipped with a CP/MAS probe for a 5 mm4, tube. The 90 ° pulse width was 5/xs. The quadrupole echo pulse sequence (90x Tl-90y--r2) was employed with 7"1 30 gs, r2 = 20 txs and 0.5 s of recycle time. 31p NMR spectra were recorded on a Chemagnetics CMX-400 spectrometer operating =
Prior to use, cytochrome c from horse heart (type VI, Sigma Chemical Co.) was converted to the fully
S. Kim et al./Journal o f Molecular Structure 441 (1998) 183-188
at 161.15MHz under proton decoupling. The decoupling strength was 50kHz. The 90 ° pulse width was 5 t~s for 31p. Chemical shifts were shown in the spectrum relative to 85% phosphoric acid.
3. Results
3.1. Binding of cytochrome c to phospholipid bilayers including cardiolipin The amount of cytochrome c bound to the lipid bilayers is shown in Table 1. Ferricytochrome c binds to cardiolipin (CL) bilayers by as much as two times as much as ferrocytochrome c does. This can be attributed to the loss of one positive formal charge at the heme and/or to a structural change of cytochrome c on reduction. Our previous results showed that ferricytochrome c binds more strongly to phosphatidylcholine(PC)/CL than to phosphatidylethanolamine (PE)/CL bilayers [23]. Since the amount of the bound cytochrome c per cardiolipin molecule is similar for PC/CL and PE/CL bilayers, the net negative charge on a liposome seems to be important for the binding of cytochrome c. Less ferricytochrome c binds to CL liposomes than to PC/CL liposomes. This suggests that the size of the binding site on the lipid bilayer for a single protein molecule is smaller than the area of the lipid bilayer covered by a protein molecule. In CL liposomes, there would be free CL molecules in the area covered by cytochrome c. They are inactive for the binding. Hence, this makes the binding efficiency of the CL liposome lower than that of the PC/CL liposome.
Table 1 The amount of cytochrome c bound to phospholipid liposomes Complexes a
Molar ratio of the bound cytochrome c to cardiolipin
CL-cyt c (ox) CL-cyt c (red) (PE/CL)-cyt c (ox) (PC/CL)-cyt c (ox)
0.067 0.033 0.094 0.093
CL, cardiolipin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; cyt c, cytochrome c; ox, oxidized form; red, reduced form. PC/CL stands for the liposomes of the binary mixture of PC and CL (4:1 w/w).
185
3.2. Deuterium quadrupole splittings of the CL glycerol backbones of the cytochrome c-lipid bilayer complexes The interactions of ferri- and ferrocytochrome c with the deuterated cardiolipin (CL*) in the bilayers were examined by 2H NMR. Fig. I(A) and (D) represent 2H NMR spectra of CL* and PC/CL* bilayers at 40°C. The assignment is given on the top of Fig. I(F) [24,26,27]. For example, 3S stands for the pro-S deuteron at the C-3 site of the glycerol backbone. The 2H NMR spectra in Fig. 1 are the superimposed powder patterns characteristic for nuclei with 1 = 1 under strong magnetic fields. The 2H NMR spectra of CL* and PC/CL* bilayers with ferri- or ferrocytochrome c are also presented in Fig. 1. The quadrupole splittings of the deuterons of CL* in the presence and absence of cytochrome c were plotted in Fig. 2 as a function of temperature. The largest error of the quadrupole splitting was -0.5 kHz for 3S deuterons. Although the changes of the quadrupole splittings are not significant, all quadrupole splittings of the glycerol deuterons of CL became smaller on binding of ferricytochrome c. This is consistent with our previous report [23]. In contrast to it, the quadrupole splittings did not change at all on binding of ferrocytochrome c. The effect of the binding of cytochrome c is similar for the single component and binary mixture bilayers. It was also reported that the quadrupole splittings of the PC component in PC/CL bilayers did not change on binding of ferricytochrome c [23].
3.3. 31p NMR spectra of the cytochrome c-lipid bilayer complexes To monitor the polymorphic structure of the samples and the effect of intermolecular interactions on the polar head groups on binding of cytochrome c, 31p NMR spectra were obtained for the same samples that were used for 2H NMR measurements. The 31p NMR spectra of CL* and PC/CL* bilayers with and without ferri- or ferrocytochrome c are shown in Fig. 3. Their powder patterns indicate that most of phospholipids are in the liquid-crystalline lamellar phase in all samples although there is a small contribution from the isotropic phase (at about 4ppm). The 31p chemical shift anisotropies ( a l l - a±) at 35°C were -31 and
186
S. Kim et al./Journal of Molecular Structure 441 (1998) 183 188
3S 2and3R !
(B)(A)~ 30
'
0 kHz
-30
30
0 kHz
-30
Fig. 1.2H NMR spectra of the deuterated glycerol backbone of cardiolipin(CL) with and without cytochrome c at 61.1 MHz and 40°C: (A) CL bilayers, (B) CL bilayers with ferricytochrome c, (C) CL bilayers with ferrocytochrome c, (D) phosphatidylcholine(PC)/CL (4:1 w/w) bilayers, (E) PC/CL (4:1 w/w) bilayers with ferricytochrome c, (F) PC/CL (4:1 w/w) bilayers with ferrocytochrome c. The assignment is given on the top of the spectrum F. Head denotes the head group of cardiolipin and the other assignments are for the prochiral deuterons of the glycerol backbone.
-43 ppm for CL* and PC/CL* bilayers, respectively. Little change was observed on binding of cytochrome c. The spectra of PC/CL* bilayers clearly consist of two axially symmetric powder patterns with different chemical shift anisotropies. The assignment of these superimposed powder patterns was reported previously [21,23,28,29]. Namely, the powder patterns with large and small chemical shift anisotropies can be ascribed to PC and CL, respectively.
Line broadening of the powder pattern of CL was observed on binding of ferri- or ferrocytochrome c. Especially, the effect of the ferrocytochrome c is significant (Fig. 3(B)). The 31p NMR spectra of the CL bilayers at 30 and 50°C are shown in Fig. 4. The linewidth became broader with an increase of temperature both for ferri- and ferrocytochrome c complexes. This fact suggests that the exchange rate between the bound and free cardiolipins is close either to the 30
-~ 30-
3S =.~_
3S
25-
25-
r.~
~. 20-
E
2 and 3R
15-
Head
2W
2 and 3R
15-
Head
.r-
IC
3'0
5b (A)
lOl
Temperature/°C
3'0
£
5b
(B)
Fig. 2. Temperature dependence of the deuterium quadrupole splittings of the deuterated glycerol backbone of cardiolipin: (A) cardiolipin bilayers, (B) phosphatidylcholine/cardiolipin (4:1 w/w) bilayers. Circles, without cytochrome c; triangles, with ferricytochrome c; crosses, with ferrocytochrome c.
S. Kim et al./Journal of Molecular Structure 441 (1998) 183-188
,
,
i
[
,
25
,
i
,
I
'
i
0
,
,
I
'
'
'
'
-25 ppm
'
'
I
'
'
25
'
'
I
i
,
0
,
,
I
'
187
'
'
-25 ppm
Fig. 3. Proton decoupled 31p NMR spectra of cardiolipin(CL) and phosphatidylcholine(PC)/CLbilayers at 161.15 MHz and 35°C: (A) CL bilayers, (B) CL bilayers with ferrocytochrome c, (C) CL bilayers with ferricytochrome c, (D) PC/CL bilayers, (E) PC/CL bilayers with ferrocytochrome c, (F) PC/CL bilayers with ferricytochrome c. chemical shift difference or to the proton decoupling strength [30]. Since the linewidth is broader for ferrothan for ferricytochrome c complexes at the same temperature, the exchange rate should be faster for the former than the latter in view o f the temperature dependence and mechanisms mentioned above. The exchange rate between the bound and free cardiolipins is much slower than the chemical shift difference or the proton decoupling strength for the ferricytochrome c - P C / C L complex at 35°C.
4. Discussion The present results showed that the only oxidized cytochrome c interacts strongly with CL in the CL
25
0 (A)
-25 ppm
25
0 (B)
bilayers and PC/CL bilayers. It was reported that ferricytochrome c specifically interacts with CL in the PC/CL bilayers but does not with CL in the PE/CL bilayers [23]. The difference was attributed to the molecular miscibility o f the two systems. Namely, while PE and CL are completely miscible, PC and CL are not [31]. In the former mixture, the strong i n t e r - p o l a r - h e a d group interaction among PE and CL, which results in a homogeneous mixing, suppresses the strong interaction between cytochrome c and CL [23]. In the mixture o f PC and CL, the presence o f microdomains was suggested [31]. However, it was not clear at that time that either CL in a PC microdomain or CL in a CL microdomain interacts with cytochrome c. Since the change o f the deuterium quadrupole splittings o f CL* on binding
,,w
-25 ppm
i , , i , , , ,
25
j,,,,
0 (C)
i,,,
-25 ppm
Fig. 4. Proton decoupled 31 P NMR spectra of CL with and without cytochrome c at 30 and 50°C: (A) cardiolipin(CL) bilayers, (B) CL bilayers with ferricytochrome c, (C) CL bilayers with ferrocytochrome c.
188
S. Kim et al./Journal of Molecular Structure 441 (1998) 183-188
of cytochrome c was larger for the single component (CL) bilayers than for the PC/CL bilayers, it can be concluded that CL in the CL microdomain should be responsible for the strong interaction with cytochrome c. In view of earlier reports, the cytochrome c-lipid interaction may affect the domain size as well. It was indicated that the binding of cytochrome c induces segregation of CL in the mixed lipid bilayers [1,32], suggesting that cytochrome c does not associate with the bulk component of the inner mitochondrial membrane, but interacts in a more specific way with cardiolipin. CL also can form a receptor surface in the inner membrane for the mitochondrial creatine kinase enzyme [ 10]. It has been proposed that the interaction of cytochrome e with lipids induce an extended lipid conformation in which the two acyl chains of a lipid point the opposite directions from the head group [33-37]. On the basis of such a model, a hydrophobic interaction between the protein and the inserted hydrocarbon chain can take place at the membrane surface without penetration of the protein into the hydrocarbon region [34,36-38]. Thus, peripheral proteins can be anchored to the membrane. In this conformation, the torsion angle among C2-C3 of the glycerol backbone of the lipid should be in the antiperiplanar range. This kind of conformation can induce a structural distortion around it. This can be one explanation for the strong interaction between ferricytochrome c and cardiolipin. Another possibility is that ferricytochrome c penetrates deeper into the membrane than ferrocytochrome c. Anyhow, the difference in the interaction between cytochrome c and lipid bilayers for the oxidized and reduced forms may play a certain role in the respiratory system in mitochondria. References [1 ] L.R. Brown, K. WiJthrich, Biochim. Biophys. Acta 468 (1977) 389. [2] M. Ryt6maa, P. Mustonen, P.K.J. Kinnunen, J. Biol. Chem. 267 (1992) 22243. [3] A. Rietveld, P. Sijens, A.J. Vekkleij, B. de Kruijff, EMBO J. 12 (1983) 907. [4l M.C. Waltham, B.A. Cornell, R. Smith, Biochim. Biophys. Acta 862 (1986) 451. [5] T.J.T. Pinheiro, A. Watts, Biochemistry 33 (1994) 2451.
[6] B. de Kruijff, P.R. Cullis, Biochirn. Biophys. Acta 602 (1980) 477. [7] R.A. Demel, W. Jordi, H. Lambrechts, H. van Damme, R. Hovius, B. de Kruijff, J. Biol. Chem. 264 (1989) 3988. [8] N.C. Robinson, F. Strey, L.H. Talbert, Biochemistry 19 (1980) 3656. [9] M. Miiller, R. Moser, D. Cheneval, E. Carafoli, J. Biol. Chem. 260 (1985) 3839. [10] D. Marsh, G.L. Powell, Bioelectrochem. Bioenerg. 20 (1988) 73. [l 1] S.B. Vik, G. Georgevich, R.A. Capaldi, Proc. Natl. Acad. Sci. USA 78 (1981) 1456. [12] H.K. Kimbelberg, Mol. Cell. Biochem. 10 (1976) 171. [13] J.M. Boggs, Membrane Fludity Biol. 2 (1983) 89. [14] P.F. Devaux, M. Seigneuret, Biochim. Biophys. Acta 822 (1985) 63. [15] J. Sedzik, A.E. Blaurock, M. H~chli, J. Mol. Biol. 174 (1984) 385. [16] W. MacNaughtan, K.A. Snook, E. Caspi, N.P. Franks, Biochim. Biophys. Acta 818 (1985) 132. [17] L. Letellier, E. Shechter, Eur. J. Biochem. 40 (1973) 507. [18] L. Mateu, F. Caron, V. Luzzati, A. Billecocq, Biochim. Biophys. Acta 508 (1978) 109. [19] T.J.T. Pinheiro, A. Watts, Biochemistry 33 (1994) 2459. [20] P.J.R. Spooner, A. Watts, Biochemistry 30 (1991) 3871. [21] P.J.R. Spooner, A. Watts, Biochemistry 31 (1992) 10129. [22] P.J.R. Spooner, A. Watts, Biochemistry 30 (1991) 3880. [23] K. Shin, T. Fujiwara, H. Akutsu, J. Mol. Struct. 355 (1995) 47. [24] W. Yoshikawa, H. Akutsu, Y. Kyogoku, Y. Akamatsu, Biochim. Biophys. Acta 944 (1988) 321. [25] H. Akutsu, Y. Suezaki, W. Yoshikawa, Y. Kyogoku, Biochim. Biophys. Acta 854 (1986) 213. [26] L.M. Strenk, P.W. Westerman, J.W. Doane, Biophys. J. 48 (1985) 765. [27] P.R. Allegrini, G. Pluschke, J. Seelig, Biochemistry 23 (1984) 6452. [28] R. Ghosh, Biochemistry 27 (1988) 7750. [29] F.M. Marassi, P.M. Macdonald, Biochemistry 30 (1991) 10558. [30] W.P. Rothwell, J.S. Waugh, J. Chem. Phys. 74 (5) (1981) 2721. [31] K. Shin, H. Maeda, T. Fujiwara, H. Akutsu, Biochim. Biophys. Acta 1238 (1995) 42. [32] G.B. Birrell, O.H. Griffith, Biochemistry 15 (1976) 2925. [33] P.K.J. Kinnunen, A. K~iv, J.Y.A. Lehtonen, M. Ryt6maa, P. Mustonen, Chem. Phys. Lipids 73 (1994) 181. [34] P.K.J. Kinnunen, Chem. Phys. Lipids 63 (1992) 251. [35] H. Hauser, 1. Pascher, S. Sundell, Biochemistry 27 (1988) 9166. [36] M. Hong, K. Schmidt-Rohr, H. Zimmermann, Biochemistry 35 (1996) 8335. [37] E. Kahana, J.C. Pinder, K.S. Smith, W.B. Gratzer, Biochem. J. 282 (1992) 75. [38] M. Ryt6maa, P.K.J. Kinnunen, J. Biol. Chem. 270 (1995) 3197.