©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reversibility of the Binding of Cytochrome c to Liposomes
IMPLICATIONS FOR LIPID-PROTEIN INTERACTIONS (*)

(Received for publication, August 29, 1994; and in revised form, November 28, 1994)

Marjatta Rytömaa Paavo K. J. Kinnunen (§)

From the Department of Medical Chemistry, Institute of Biomedicine, P.O. Box 8 (Siltavuorenpenger 10 A), FIN-00014 University of Helsinki, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Studies on the membrane binding of cytochrome c revealed liposome-associated and soluble cytochrome c not to be in rapid equilibrium. In brief, cytochrome c attached to pyrene phospholipid-labeled, fluorescent liposomes containing either 17.6 mol % cardiolipin (CL) or 30 mol % egg phosphatidylglycerol (PG) is practically not at all or very slowly, respectively, detached by a subsequently added excess (up to 20-fold) of nonlabeled liposomes containing these acidic lipids. Cytochrome c was fully dissociated from PG-containing liposomes by increasing the ionic strength by NaCl, whereas dissociation from CL-containing membranes was less complete, presumably because of the scavenging of the protein within inverted intramembrane micelles. Importantly, the apparent irreversibility of the binding of cytochrome c to liposomes is strongly dependent on the structure of the acidic phospholipid. Cytochrome c bound to lyso-PG/PC liposomes could be dissociated with an excess of nonlabeled PG-containing liposomes. Cytochrome c was also efficiently bound to membranes containing the negatively charged dicetylphosphate yet could be readily dissociated by nonlabeled PG-containing liposomes. We conclude both proper geometry of the phosphate group and the presence of two acyl chains to be required for the tight binding of cytochrome c to acidic phospholipids. These data provide evidence for the membrane association of cytochrome c by an acidic phospholipid in the extended conformation (Kinnunen, P. K. J., Kõiv, A., Lehtonen, J. Y. A., Rytömaa, M., and Mustonen, P. (1994) Chem. Phys. Lipids 73, 181-207) in which one of the acyl chains of the lipid becomes accommodated within a hydrophobic cavity of the protein. Based on the crystal structure of cytochrome c we putatively assign the invariant Asn-52 (horse heart cytochrome c) as the site liganding the protonated phosphate of the lipid, whereas Lys-72 and -73 should bind the deprotonated form.


INTRODUCTION

Peripheral membrane interactions have been widely studied and include the lipid association of proteins such as cytochrome c(1, 2, 3, 4, 5, 6, 7, 8, 9) and protein kinase C (10) as well as nucleic acids (11, 12) and drugs such as adriamycin(13, 14) . The lipid requirements for the membrane association of cytochrome c and protein kinase C are fairly well understood. Accordingly, cytochrome c binds to membranes containing acidic phospholipids and cardiolipin either as such or complexed with cytochrome c oxidase is likely to provide its binding site at the inner mitochondrial membrane(15, 16) . For the activation of protein kinase C, diacylglycerol is needed in addition to phosphatidylserine(17) .

Cytochrome c has been considered as a paradigm for electrostatically interacting peripheral membrane proteins since it is dissociated from lipid membranes by increasing ionic strength(18, 19, 20) . However, also hydrophobic interactions have been reported to be involved in the membrane association of cytochrome c(21) . The binding of cytochrome c to liposomes has been shown to induce conformational alterations in the protein as well as in the phospholipids(22, 23, 24, 25, 26, 27, 28, 29, 30, 31) . We have previously suggested that there are two distinct acidic phospholipid binding sites in cytochrome c and have tentatively nominated these as A- and C-site, for anion and cardiolipin binding, respectively(20, 32) . These putative sites are characterized as follows. The A-site interacts with anions such as deprotonated acidic phospholipids, Cl, and nucleotides. Accordingly, the binding of cytochrome c to liposomes by the A-site is likely to result from electrostatic interactions between the protein and acidic phospholipids and can be reversed with increasing ionic strength and anions. In contrast the C-site presumably involves hydrogen bonding between cytochrome c and protonated acidic phospholipids, and the protein is not dissociated with nucleotides or increasing ionic strength. However, sufficiently high ionic strength results in the deprotonation of acidic phospholipids and thus causes the dissociation of cytochrome c. The nature of the membrane association of cytochrome c can be controlled by factors affecting the protonation of the acidic phospholipids, i.e. pH(20) , ionic strength, or surface charge density, that is the amount of acidic phospholipids, in liposomes(32) .

In the course of our studies we observed that once cytochrome c became bound to pyrene-labeled, fluorescent liposomes via either its A-site or C-site it was no longer dissociated by nonfluorescent liposomes added afterwards. This phenomenon and its dependence on the acidic phospholipid structure are reported here. Formation of the hexagonal H phase as a consequence of the association of cytochrome c to cardiolipin-containing membranes has been demonstrated(33) . Therefore, although CL (^1)is likely to constitute the physiological binding site for cytochrome c we also studied the reversibility of the association of cytochrome c with PG-containing liposomes in order to avoid ambiguities in the interpretation of the results arising because of the formation of H phase. The experiments were conducted using large unilamellar liposomes containing either 17.6 mol % CL, or 30 mol % PG, lyso-PG, or dicetylphosphate. Under these conditions the A-site is functional, and cytochrome c is detached from the liposomes by ATP.


EXPERIMENTAL PROCEDURES

Materials

1-Palmitoyl-2-[10-(pyren-1-yl)]decanoyl-sn-glycero-3phosphocholine (PPDPC) was purchased from K & V Bioware (Espoo, Finland). Horse heart cytochrome c (type VI, oxidized form), egg PC, egg PG, dicetylphosphate (DCP), lyso-PC, DPPG, DPPC, and cholesterol were from Sigma, and 1-palmitoyl-sn-glycero-3-phosphoglycerol (lyso-PG) was from Alexis (Switzerland). No impurities were detected in the above lipids upon thin layer chromatography on silicic acid using chloroform/methanol/water/ammonia (65:20:2:2, v/v) as the solvent system and examination of the plates for pyrene fluorescence or after iodine staining.

Preparation of Liposomes

Lipids were dissolved in chloroform and mixed in this solvent to obtain the desired compositions. PPDPC was used as the fluorescence energy donor, and its content in labeled liposomes was 1 mol %. After mixing the lipids, the solvent was removed under a stream of nitrogen. The lipid residue was subsequently maintained under reduced pressure for at least 2 h and then hydrated in 20 mM Hepes, 0.1 mM EDTA, pH 7.0, at room temperature to yield a lipid concentration of 1-5 mM. To obtain unilamellar vesicles, the hydrated lipid mixtures were extruded with a LiposoFast small volume homogenizer (Avestin, Ottawa, Canada). Samples were subjected to 19 passes through two polycarbonate filters (100 nm pore size, Nucleopore, Pleasanton, CA) installed in tandem(34) . Minimal exposure of the lipids to light was ensured throughout the above procedure. Subsequently, the liposome solution was divided into proper aliquots and diluted with the buffer to a final lipid concentration of 10.6-12.5 µM, as indicated, of the pyrene-containing liposomes. In order to allow for comparison between CL and PG the stoichiometries of these lipids in liposomes were adjusted to maintain the final concentration of phosphate groups in liposomes constant. In other words CL with two protonating phosphates and four acyl chains was regarded equivalent to two PGs. Accordingly, the lipid concentration in Fig. 1B is given in terms of the amount of phosphate.


Figure 1: A, binding of cytochrome c to PC (10.6 µM total phospholipid) liposomes containing 1 mol % PPDPC and 17.6 mol % CL (, bullet, , +). The open symbols show the binding of cytochrome c to PPDPC/CL/PC liposomes mixed with nonfluorescent liposomes (17.6 mol % CL in PC) prior to the addition of the protein. The molar ratios of pyrene-labeled/nonlabeled liposomes were 1/1 (box), 1/5 (circle), 1/10 (Delta), 1/20 (down triangle). B, effect caused by the addition of nonlabeled liposomes containing 17.6 (, +), 33.3 (bullet), or 100 () mol % CL subsequent to the binding of cytochrome c to labeled, PPDPC-containing liposomes. The upward arrow indicates the dissociation of cytochrome c by 150 mM NaCl. The lipid concentration indicated on the x-axis in panel B represents the concentration of phosphate groups. For the graph labeled by (+), cytochrome c was added only up to a concentration of 123 nM (panel A, downward arrow), after which nonlabeled PC liposomes containing 17.6 mol % CL were added. Cyt, cytochrome.



Binding of Cytochrome c to Liposomes

The lipid binding of cytochrome c was assessed essentially as described previously (20, 32) by monitoring Perrin-Förster resonance energy transfer (35, 36) between pyrene-labeled lipids and the heme of cytochrome c. In brief, excitation at 344 nm and emission at 394 nm were selected with monochromators and using 1- and 16-nm slits, respectively, of our SLM 4800S spectrofluorometer. Two ml of liposome solution was placed into a magnetically stirred four-window cuvette in a holder thermostated with a circulating water bath at 25 °C. Ten-µl aliquots of a 5 or 10 µM solution of cytochrome c were subsequently added, and the quenching of pyrene fluorescence by the heme of cytochrome c was observed(7, 20, 32) . Thereafter, when indicated, proper aliquots of 1-5 mM nonlabeled liposome solutions were included. Unless otherwise stated changes in fluorescence were allowed to stabilize for approximately 40 s, after which the intensity of pyrene monomer fluorescence was recorded. Because of the low concentrations of both lipids and cytochrome c utilized, minimal interference by the inner filter effect is expected. This is also evident from the efficient reversal of fluorescence quenching caused by the addition of 150 mM NaCl, which practically completely detaches cytochrome c from PG-containing liposomes, evident as full recovery of the original fluorescence intensity (Fig. 4). As judged from its absorption spectra, the cytochrome c used was mainly in the oxidized form. The merits as well as limitations of the use of pyrene-labeled lipids in energy transfer measurements have been discussed elsewhere(13, 20, 37, 38) .


Figure 4: A, binding of cytochrome c to PC liposomes (12.5 µM total phospholipid) containing 1 mol % PPDPC and 30 mol % PG (, bullet, , +). The open symbols show the binding of cytochrome c to PPDPC/PG/PC liposomes mixed with nonlabeled (30 mol % PG in PC) prior to the addition of the protein. The molar ratios of pyrene-labeled/nonlabeled liposomes were 1/1 (box), 1/5 (circle), 1/10 (Delta), and 1/20 (down triangle). B, the addition of nonlabeled liposomes containing 30 (, +), 50 (bullet), or 100 () mol % PG subsequent to the addition of cytochrome c. The upward arrow indicates the dissociation of cytochrome c by 150 mM NaCl. Similarly to Fig. 1for the graph labeled (+), cytochrome c was added only up to a concentration of 123 nM (panel A, downward arrow), after which nonlabeled liposomes (30 mol % PG in PC) were added. Cyt, cytochrome.




RESULTS

Reversibility of Cytochrome c Binding to CL or PG-containing Liposomes

In the course of our studies on the binding of cytochrome c to liposomes we observed that once bound the protein did not readily equilibrate with a subsequently added excess of identical liposomes. This is illustrated in Fig. 1using liposomes containing 17.6 mol % CL. In brief, increasing [cytochrome c] results in a progressive quenching of fluorescence caused by the membrane association of cytochrome c, which allows for an efficient Förster type dipole-dipole coupling between the excited pyrene and the heme of cytochrome c. As expected, when increasing concentrations of nonlabeled liposomes are present prior to the addition of cytochrome c, the partitioning of cytochrome c to pyrene-containing liposomes becomes reduced and less efficient fluorescence quenching is evident (Fig. 1A). However, if nonlabeled liposomes are added after the association of cytochrome c to pyrene-labeled liposomes has been first allowed to take place, very little dissociation of cytochrome c from the fluorescent liposomes is observed (Fig. 1B). The maximal extent of detachment was essentially constant and independent from the amount of nonlabeled liposomes. However, if nonlabeled liposomes are added at a point where the low affinity binding of cytochrome c to liposomes has not commenced, essentially no detachment of cytochrome c from liposomes is seen even in the presence of a 20-fold excess of nonlabeled liposomes (Fig. 1). The distribution of cytochrome c added to a mixture of pyrene-labeled and nonlabeled liposomes is also dependent on the amount of acidic phospholipids, i.e. surface charge density of the latter (Fig. 2). Accordingly, cytochrome c has a higher affinity for liposomes containing more acidic phospholipids and thus also having a higher surface charge density, thus probably reflecting higher affinity of cytochrome c to protonated acidic phospholipids(32) . The apparent non-equilibrium described above is not limited by slow kinetics. Essentially no changes were seen in fluorescence upon incubation of membrane-associated cytochrome c with a 20-fold excess of subsequently added nonlabeled liposomes for up to 6 h (Fig. 3). -Cytochrome c has been shown to induce the formation of hexagonal phase H as a consequence of its association with CL(33) . We have previously suggested this to result in the translocation and trapping of cytochrome c into the liposomes, causing the incomplete dissociation of cytochrome c from CL-containing liposomes by NaCl(32) , as shown in Fig. 1B. In order to avoid interference caused by the cytochrome c-induced formation of inverted micelles in CL-containing liposomes we repeated the above experiments using PG as the acidic phospholipid ( Fig. 4and Fig. 5). Except for the nearly complete detachment of cytochrome c by salt from PG-containing vesicles (Fig. 4B) similar results were obtained as described above for CL. Yet, whereas the tight association of cytochrome c with CL appears to be practically irreversible, a slow dissociation of cytochrome c from labeled PG/PC liposomes by nonlabeled PG/PC liposomes is seen (Fig. 3). However, even after 6 h this reversal is incomplete. Notably, when cytochrome c was bound to pyrene labeled liposomes containing 30 mol % PG the dissociation by PC liposomes containing either 30 mol % PG or 17.6 mol % CL was similar (data not shown). The low affinity binding of cytochrome c to pyrene-labeled PG/PC liposomes could also be reversed with an excess of nonlabeled PC liposomes (data not shown). Likewise, during a 5-h incubation of cytochrome c bound to labeled PG/PC liposomes with a 20-fold excess of nonlabeled PC liposomes only an insignificant increase in the fluorescence intensity was observed (Fig. 3).


Figure 2: Lack of reversal of the membrane association of cytochrome c (244 nM) caused by subsequently added nonlabeled liposomes containing 17.6 (), 33.3 (bullet), or 100 mol % () CL. The open symbols show the effect of the same concentrations of nonlabeled liposomes added prior to the addition of cytochrome c. The data were taken from Fig. 1and other similar measurements. Lipid concentration in fluorescent liposomes was 10.6 µM.




Figure 3: Time course of the detachment of cytochrome c from pyrene-labeled liposomes by subsequently added nonlabeled liposomes. Cytochrome c (244 nM) was allowed to bind to liposomes (12.5 µM phosphate) containing in addition to PPDPC and PC either 30 mol % PG (, ) or 17.6 mol % CL (bullet), after which a 20-fold excess (i.e. 250 µM phosphate) of nonlabeled PG/PC (30 mol % PG) (), PC (), or CL/PC (17.6 mol % CL) (bullet) liposomes was included. Arrows indicate the dissociation of cytochrome c by 150 mM NaCl.




Figure 5: The reversal of the membrane association of cytochrome c (244 nM) caused by nonlabeled liposomes containing 30 (, box), 50 (bullet, circle), or 100 mol % (, ) PG. The closed symbols show the effect of nonlabeled liposomes added subsequent to the addition of cytochrome c, and the open symbols show the effect of nonlabeled liposomes added prior to the addition of cytochrome c. The data were taken from Fig. 4and other similar measurements. The lipid concentration of fluorescent liposomes was 12.5 µM.



The above experiments utilized liposomes in the fluid, liquid crystalline state. The tight binding of cytochrome c to liposomes containing 30 mol % egg PG was not dependent on the membrane phase state, however. Accordingly, nonlabeled liposomes did not dissociate cytochrome c from PPDPC/DPPG/DPPC liposomes below their main phase transition temperature at 41 °C. Likewise, the presence of up to 50 mol % (from the final lipid concentration) cholesterol did not affect the apparently irreversible binding of cytochrome c described above (data not shown).

Binding of Cytochrome c to Lyso-PG/PC Liposomes

To study the importance of the acyl chains in the negatively charged phospholipid for the membrane association of cytochrome c we gradually substituted lyso-PG for PG in liposomes. More specifically, the content of acidic phospholipid was maintained constant at 30 mol % while the lyso-PG/PG ratio was varied. Concomitantly, in order to avoid major changes in membrane curvature the content of lysophospholipids was also maintained constant at 30 mol % by adding lyso-PC as the amount of lyso-PG decreased. The efficiency of fluorescence quenching by cytochrome c increases gradually when substituting lyso-PG by increasing amounts of PG in the liposomes, thus indicating more efficient binding of cytochrome c to liposomes containing an acidic phospholipid with two acyl chains (Fig. 6A). Interestingly, the binding of cytochrome c to labeled lyso-PG/PC liposomes was rapidly and effectively reversed by the subsequent addition of PG/PC liposomes (Fig. 6B). However, this reversal becomes progressively reduced upon decreasing the lyso-PG/PG ratio in the fluorescent liposomes. Cytochrome c was also detached from labeled lyso-PG/PC liposomes by a large excess of nonlabeled PC liposomes (Fig. 7).


Figure 6: A, binding of cytochrome c to liposomes containing in addition to 1 mol % of PPDPC 30 mol % lyso-PG (); 20 mol % lyso-PG, 10 mol % PG, 10 mol % lyso-PC (bullet); 10 mol % lyso-PG, 20 mol % PG, 20 mol % lyso-PC (); or 30 mol % PG, 30 mol % lyso-PC (+), the balance in each case being PC. B, the dissociation of cytochrome c by nonlabeled liposomes containing 30 mol % PG in PC. The arrow indicates the dissociation of cytochrome c by 150 mM NaCl. Cyt, cytochrome.




Figure 7: A, binding of cytochrome c to labeled (1 mol % of PPDPC) PC liposomes containing 30 mol % lyso-PG (bullet). B, the dissociation of cytochrome c by nonlabeled PC liposomes. Cyt, cytochrome.



To exclude possible interference caused by transfer of lyso-PG between liposomes we mixed lyso-PG/PPDPC/PC (30/5/65 molar ratio) with a 5-fold molar excess of PG/PC (30/70 molar ratio) liposomes. If lyso-PG would equilibrate between the two liposome populations this would result in an enrichment of the fluorescent lipid PPDPC. This in turn would cause an increase in I(e)/I(m), i.e. the ratio of excimer/monomer fluorescence emission intensities(38) . However, practically no changes in I(e)/I(m) were observed within a 30-min incubation, indicating that the spontaneous transfer of lyso-PG between liposomes under these conditions is slow. Notably, transfer of lyso-PG in the above system would also involve curvature changes, which are counteracted by the bending rigidities of the membranes. We also investigated the possibility that the above results (Fig. 6) could be explained by a rapid equilibration of the lyso-PG-cytochrome c complex between labeled and nonlabeled liposomes. If this was the case the extent of fluorescence quenching caused by the binding of cytochrome c to a 1/1 (molar ratio) mixture of two liposome populations of either (a) labeled lyso-PG/PC liposomes and nonlabeled PC liposomes or (b) labeled PC liposomes and nonlabeled lyso-PG/PC liposomes should be identical. However, compared with cytochrome c bound to labeled lyso-PG/PC liposomes the quenching of pyrene fluorescence decreases only slightly (from 49 to 41%) when cytochrome c is bound to a mixture of labeled lyso-PG/PC and nonlabeled PC liposomes, whereas little binding to labeled PC vesicles is observed in the presence of nonlabeled lyso-PG/PC liposomes (19% quenching, data not shown).

Binding of Cytochrome c to Dicetylphosphate-containing Liposomes

Cytochrome c avidly binds to liposomes containing 30 mol % of DCP as the acidic phospholipid. Although fluorescence quenching caused by the heme protein is not as effective as for PG- or CL-containing liposomes, it is comparable with liposomes containing lyso-PG (Fig. 8A). Notably, the binding of cytochrome c to these liposomes is reversible, and the bound protein is readily dissociated from PPDPC/DCP/PC liposomes when nonlabeled liposomes are being added (Fig. 8B), thus suggesting that cytochrome c becomes rapidly equilibrated between pyrene-labeled and nonlabeled liposomes. Complete dissociation of cytochrome c from labeled DCP/PC vesicles is observed following the addition of an equal concentration of nonlabeled liposomes containing either 30 or 100 mol % PG, thus indicating a much higher affinity of cytochrome c for egg PG than DCP. However, not all of the surface bound cytochrome c is dissociated with nonlabeled liposomes since the inclusion of 150 mM NaCl causes a further small increase in fluorescence. Because of the molecular geometry of DCP one might anticipate this lipid to have some tendency to form the H phase(40) , similarly to CL(33) .


Figure 8: A, binding of cytochrome c to PC liposomes containing 1 mol % PPDPC and 30 mol % DCP (three identical measurements are shown). B, the addition of nonlabeled liposomes containing in addition to PC, 30 mol % dicetylphosphate (), 30 mol % PG (bullet), or 100 mol % PG (). The arrow indicates the dissociation of cytochrome c by 150 mM NaCl. Cyt, cytochrome.




DISCUSSION

We have recently shown that the peripheral binding of cytochrome c to membranes also involves nonelectrostatic interactions (20) dependent on the protonation of acidic phospholipids(32) . In brief, we proposed two distinct acidic phospholipid binding sites in cytochrome c, the C-site recognizing protonated and the A-site recognizing deprotonated acidic phospholipids. Dissociation of cytochrome c attached to membrane via its A-site, i.e. from liposomes containing 30 mol % PG can be induced with increasing ionic strength as well as by the addition of ATP. The electrostatically membrane-associated cytochrome c should be in equilibrium with the protein in solution, thus resulting in the reversal of fluorescence quenching caused by the addition of an excess of nonlabeled liposomes. However, as shown in the present work, cytochrome c bound via its A-site to CL- or PG-containing liposomes is poorly detached by a 20-fold excess of subsequently added nonlabeled liposomes. We have previously noted the biphasic association of cytochrome c with acidic phospholipids containing liposomes and have suggested saturation of the high affinity binding sites to be followed by a loose, less specific association of cytochrome c to the surface of liposomes(20) . It appears that only the latter, less tightly bound cytochrome c appears to be readily removable by subsequently added nonfluorescent liposomes. Dissociation of cytochrome c from membranes containing PG is immediately (within a few seconds) induced by increasing ionic strength. Accordingly, the apparent irreversibility of cytochrome c binding to liposomes is unlikely to involve penetration of the protein into the lipid bilayer. In this context it is worth noticing that highly analogous behavior has been described for the membrane-bound protein kinase C, which does not equilibrate with subsequently added liposomes while rapid dissociation of protein kinase C is induced by chelating Ca with EGTA(41) . The inability of excess nonlabeled liposomes to dissociate membrane bound cytochrome c could result from either a highly cooperative membrane association or very slow dissociation kinetics of the cytochrome c-lipid complex, thus favoring the membrane-bound form of the protein. However, it is difficult to reconcile how the latter could be compatible with an electrostatic membrane association of cytochrome c.

The binding of cytochrome c to DCP- or lyso-PG-containing liposomes is almost identical, in keeping with the electrostatic interaction of cytochrome c with these lipids. Likewise, cytochrome c is effectively displaced from these lipids by ATP, in keeping with their acidic headgroup interacting with the A-site of the protein (data not shown). In DCP the two alkyl chains are attached directly to the phosphate group. The rapid dissociation of cytochrome c from DCP-containing liposomes by PG/PC liposomes reveals the importance of the correct glycerophospholipid structure for the tight and apparent nonequilibrium membrane association of the protein. Another structural alteration in the acidic lipid resulting in the rapid detachment of bound cytochrome c by PG/PC liposomes is substitution of lyso-PG for PG in the fluorescent vesicles. Because of the negative charge of lyso-PG simultaneous substitution of lyso-PG for lyso-PC is somewhat inadequate in maintaining the curvature of the liposomes. However, since it was desirable to maintain a constant negative surface charge density lyso-PC provides the best available control for the curvature changes caused by lyso-PG. In addition, the dissociation of cytochrome c from lyso-PG-containing liposomes by nonlabeled liposomes is unlikely to be caused by increased membrane curvature by this lyso-component since the efficiency of cytochrome c induced fluorescence quenching actually slightly increases when lyso-PC is present in PG-containing liposomes. Accordingly, if an increase in membrane curvature increases the binding of cytochrome c to liposomes the increase should be even greater when liposomes contain lyso-PG. However, upon gradually substituting lyso-PG for PG the extent of fluorescence quenching caused by the membrane association of cytochrome c decreases, and the detachment of cytochrome c from labeled liposomes by nonlabeled liposomes becomes progressively increased. Taken together, two requirements for the tight, slowly equilibrating membrane association of cytochrome c emerge. These are (a) the presence of two acyl chains in the acidic diacylphospholipid and (b) the correct structure of its phosphate group.

We have recently proposed a novel mechanism for the peripheral attachment of proteins to lipid membrane surfaces(42) , based on the so-called extended lipid conformation (43) in which the two acyl chains of a lipid are pointed to opposite directions from the headgroup. This requires the torsion angle between the glycerol backbone carbon atoms C-2 and C-3 to be in the antiperiplanar range, as seen with NMR(44) . The extended lipid anchorage of peripheral proteins to membranes involves the accommodation of one of the lipid acyl chains within a hydrophobic cavity in the protein while the other chain remains in the membrane. This configuration thus results in a hydrophobic interaction between the protein and the lipid surface without penetration of the protein into the membrane hydrocarbon region(42) . Interestingly, Kahana et al.(45) reported that spectrin and some other red cell membrane cytoskeletal proteins have fatty acid binding sites, and they suggested this property to be a general feature of peripheral membrane proteins. Importantly, there is in cytochrome c a channel with an unknown function, lined by hydrophobic amino acid residues and leading from the surface of the protein into the heme crevice(46) . One side of the opening of this channel on the protein surface is constituted by a highly conserved cluster of positively charged lysines. This cluster is opposed on the other side by the invariant asparagine (Asn-52 in horse cytochrome c)(46) . In light of the structural studies on cytochrome c and the features of the lipid association of this protein we are suggesting the putative mechanism illustrated in Fig. 9for the binding of deprotonated and protonated acidic phospholipids to the A- and C-site, respectively. In both mechanisms one acyl chain would be accommodated within the hydrophobic channel. The A-site would involve electrostatic interactions of the deprotonated phosphate with the conserved lysines at positions 72 and 73. The C-site in turn would involve the invariant Asn-52 forming two hydrogen bonds with the protonated phosphate (Fig. 10). For CL with two phosphates, both polar interactions, i.e. docking of one protonated phosphate moiety to Asn together with a simultaneous electrostatic attachment of the other, deprotonated phosphate to the lysines, are possible. Hydrogen bonding by Asn is greatly enhanced in a hydrophobic environment, i.e. in a low dielectric medium(47) . The enhanced ability of this Asn to form hydrogen bonds is also evident from studies on the structural water molecules in cytochrome c(48) . Accordingly, one of the water molecules (Wat-1) is hydrogen bonded to Asn-52, Tyr-67, and the carbonyl of Ile-75(48) . This structure of the C-site would also be compatible with the close vicinity of one of the CL phosphates to the heme moiety(29, 30) . In this context it is of interest that Asn and Gln are abundant in the interfacial regions of transmembrane proteins, adjacent and in close contact with the headgroups of membrane lipids(49) . Examples of such proteins are Escherichia coli lactose permease (50) and the platelet-activating factor receptor(51) . Accordingly, it is tempting to suggest that Asn and Gln would be generally involved in liganding protonated acidic phospholipids to the boundary of transmembrane proteins.


Figure 9: Model for the binding of cytochrome c to acidic phospholipids. Top, the A-site binding of cytochrome c to PG. Bottom, the C-site binding of cytochrome c to PG. See ``Discussion'' for details.




Figure 10: Hydrogen bonding between Asn and protonated phosphate of an acidic phospholipid. Rrepresents the glycerol backbone containing two acyl chains. R represents the polar head group glycerol in PG.



The depicted mechanism is compatible with the available data on cytochrome c-lipid association, such as the involvement of hydrophobic interactions in the absence of penetration of cytochrome c into lipid monolayers(52) , as well as the depletion of acyl chains from the lipid monolayer in contact with cytochrome c demonstrated by x-ray studies(53) . Catalytic hydrogenation of the unsaturated lipids of mitochondria reveals that in the presence of membrane proteins the acyl chain double bonds of CL are less accessible to reduction than those of other mitochondrial lipids (54) . However, differences between the various lipids were abolished in the absence of membrane proteins. This could easily be understood if some of the acyl chains in CL are accommodated within the hydrophobic channel of cytochrome c and perhaps also other mitochondrial proteins.

For the A- and C-site interactions the rate-limiting contribution would be ionic and hydrogen bonding, respectively. Accordingly, the former interaction would be competitively blocked by, for instance, ATP(20) , whereas the latter would be efficiently controlled by the protonation of the phosphate head group of the acidic lipids, further influenced by factors such as pH, surface charge density, and membrane potential(32, 55) . The rate constant for the association of cytochrome c to either PG- or CL-containing surfaces (k) should greatly exceed the value for the rate constant for detachment (k). This difference between the rate constants should arise because of the involvement of hydrophobic interactions. It is possible that an electrostatic interaction of cytochrome c with liposomes is initially required for the binding of the protein to the liposome surface in the right orientation and perhaps also for the opening of the cavity of the protein. Conformational changes and destabilization of protein structure (27) have been reported as a consequence of cytochrome c-lipid interactions(25, 26, 27, 28, 29, 30, 31) . The mechanism for cytochrome c lipid interactions illustrated here could also be involved in the membrane association of other proteins such as protein kinase C, CTP:phosphocholine cytidyltransferase, and annexins(42) . For example, in the case of annexin V an interaction between the phosphate group and the protein and a hydrophobic interaction between the sn-2 acyl chain and the protein seem to be involved(56) .


FOOTNOTES

*
Financial support was obtained from the Finnish Academy, Sigrid Juselius Foundation, Biocenter Helsinki, and the 350 Years Anniversary Foundation of the University of Helsinki. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 358-0-1918276.

(^1)
The abbreviations used are: CL, cardiolipin; DCP, dicetylphosphate; DPPC, dipalmitoylphosphatidylcholine; DPPG dipalmitoylphosphatidylglycerol; PC, egg phosphatidylcholine; PG, egg phosphatidylglycerol; lyso-PC, 1-palmitoyl-sn-glycero-3-phosphocholine; lyso-PG, 1-palmitoyl-sn-glycero-3-phosphoglycerol; PPDPC, 1-palmitoyl-2-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine; RFI, relative fluorescence intensity.


ACKNOWLEDGEMENTS

We thank Drs. Pekka Mustonen and Jukka Lehtonen for rewarding discussions on the topic of this paper. The technical assistance of Birgitta Rantala is appreciated.


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