Cardiolipins and biomembrane function.

Evidence is discussed for roles of cardiolipins in oxidative phosphorylation mechanisms that regulate State 4 respiration by returning ejected protons across and over bacterial and mitochondrial membrane phospholipids, and that regulate State 3 respiration through the relative contributions of proteins that transport protons, electrons and/or metabolites. The barrier properties of phospholipid bilayers support and regulate the slow proton leak that is the basis for State 4 respiration. Proton permeability is in the range 10(-3)-10(-4) cm s-1 in mitochondria and in protein-free membranes formed from extracted mitochondrial phospholipids or from stable synthetic phosphatidylcholines or phosphatidylethanolamines. The roles of cardiolipins in proton conductance in model phospholipid membrane systems need to be assessed in view of new findings by Hübner et al. [313]: saturated cardiolipins form bilayers whilst natural highly unsaturated cardiolipins form nonlamellar phases. Mitochondrial cardiolipins apparently participate in bilayers formed by phosphatidylcholines and phosphatidylethanolamines. It is not yet clear if cardiolipins themselves conduct protons back across the membrane according to their degree of fatty acyl saturation, and/or modulate proton conductance by phosphatidylcholines and phosphatidylethanolamines. Mitochondrial cardiolipins, especially those with high 18:2 acyl contents, strongly bind many carrier and enzyme proteins that are involved in oxidative phosphorylation, some of which contribute to regulation of State 3 respiration. The role of cardiolipins in biomembrane protein function has been examined by measuring retained phospholipids and phospholipid binding in purified proteins, and by reconstituting delipidated proteins. The reconstitution criterion for the significance of cardiolipin-protein interactions has been catalytical activity; proton-pumping and multiprotein interactions have yet to be correlated. Some proteins, e.g., cytochrome c oxidase are catalytically active when dimyristoylphosphatidylcholine replaces retained cardiolipins. Cardiolipin-protein interactions orient membrane proteins, matrix proteins, and on the outerface receptors, enzymes, and some leader peptides for import; activate enzymes or keep them inactive unless the inner membrane is disrupted; and modulate formation of nonbilayer HII-phases. The capacity of the proton-exchanging uncoupling protein to accelerate thermogenic respiration in brown adipose tissue mitochondria of cold-adapted animals is not apparently affected by the increased cardiolipin unsaturation; this protein seems to take over the protonophoric role of cardiolipins in other mitochondria. Many in vivo influences that affect proton leakage and carrier rates selectively alter cardiolipins in amount per mitochondrial phospholipids, in fatty acyl composition and perhaps in sidedness; other mitochondrial membrane phospholipids respond less or not at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.     

Calorimetric studies of cytochrome oxidase-phospholipid interactions

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.     

Activation of beef-heart cytochrome c oxidase by cardiolipin and analogues of cardiolipin

Beef-heart cytochrome c oxidase lacking endogenous lipids can be prepared by cholate-mediated exchange with dimyristoylphosphatidylcholine (Powell, G. L., Knowles, P. F. and Marsh, D. (1985) Biochim. Biophys. Acta 816, 191-194). These preparations retained practically no endogenous cardiolipin (less than 0.19 mol cardiolipin per mol of oxidase) but in Tween 80 they retained unaltered electron transport activity. Resupplementation of the dimyristoylphosphatidylcholine-substituted cytochrome oxidase with cardiolipin and cardiolipin analogues with different numbers of acyl chains or with a methylated headgroup enhanced the activity of the reconstituted enzyme to an extent dependent on the structure of the cardiolipin derivative. The Eadie-Hofstee plot showed biphasic kinetic behavior for all reconstituted preparations, even those completely lacking cardiolipin. This biphasic substrate dependence of the kinetics was simulated using the model of Brzezinski, P. and Malmstr?m, B. G. (Proc. Natl. Acad. Sci. USA 83 (1986) 4282-4286), which implicates two interconverting enzyme conformations in the proton transport step. The activation of cytochrome c oxidase by the cardiolipin analogues could be explained in terms of an electrostatic enhancement of the surface concentrations of both cytochrome c and protons, and a facilitated interconversion between the two enzyme conformations.     

Cytochrome c specifically induces non-bilayer structures in cardiolipin-containing model membranes

(1) The effect of cytochrome c addition on the phospholipid structure of liposomes composed of cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidylcholine or phosphatidylethanolamine in a pure form or in mixtures was investigated by 31P-NMR and freeze-fracture techniques. (2) Cytochrome c specifically induces the hexagonal Hii phase and possibly an inverted micellar structure of part of the phospholipids in cardiolipin-containing model membranes. (3) These results are compared with the effect of Ca2+ on cardiolipin and are discussed in relation to the structure and function of the inner mitochondrial membrane.     

Spin-label study of the relation between enzymatic activity and lipid-protein organization in reconstituted cytochrome c oxidase.

Lipid-depleted cytochrome c oxidase (EC 1.9.3.1) containing less than 20 microgram lipids per milligram protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and (or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipds such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology, and a high specific activity, was examined by electron spin resonance spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid - containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Our results have established that aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.     

Lipid regulation of bovine cytochrome P450(11) beta activity

Purified bovine adrenocortical cytochrome P450(11) beta has been reconstituted into phospholipid vesicles using a detergent dialysis procedure. Using this reconstituted system, we have examined the effect of changes in the fatty acyl substituents of the lipids on the catalytic activity of the enzyme. The studies reported here show that cytochrome P450(11) beta exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450scc. Cytochrome P450(11) beta displays maximal activity in lipid vesicles composed of saturated lipids, such as dipalmitoyl and dimyristoyl phosphatidylcholines, with turnover numbers ranging from 35 to 60 min-1. Incremental increases of phospholipids such as diphytanoyl and dioleoyl phosphatidylcholines result in a progressive inhibition of 11 beta hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in Vmax accompanied by modest changes in Km for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450scc, is a potent inhibitor of the 11 beta hydroxylase activity of cytochrome P450(11) beta, with half maximum inhibition observed in vesicles containing 4-5 mol% diphosphatidyl glycerol. Kinetic analysis demonstrates that this inhibition by diphosphatidyl glycerol is reflected in both a decrease in Vmax and relatively large increases (up to sevenfold) in Km for the steroid substrate. These effects on the 11 beta hydroxylase activity may have important implications for the in vivo regulation of not only the 11 beta hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.     

Relationship between losses in cytochrome oxidase activity and peroxidation of monosaturated phosphatidylcholines and phosphatidylethanolamines.

Incubation of inner mitochondrial membranes from rat liver in the presence of inducers of peroxidation reactions, such as ascorbate or cysteine, produced a large loss in cytochrome oxidase activity parallel to the disappearance of phosphatidylcholine and phosphatidylethanolamine molecular species, which contained a saturated and an unsaturated fatty acid. The loss in enzyme activity was unrelated to alterations in other species of these phospholipids or other ones. These results may reflect the existence of specific associations within the membrane between cytochrome oxidase and monosaturated phosphatidylcholines and/or phosphatidylethanolamines.     

Influence of detergent polar and apolar structure upon the temperature dependence of beef heart cytochrome c oxidase activity

The temperature dependence of lipid-depleted beef heart cytochrome c oxidase activity was studied in a series of chemically homogeneous detergents. The detergents that were tested included C10 to C18 maltosides, C8 to C12 glucosides, C8 to C16 Zwittergents, and C12 poly(oxyethylene) ethers. The observed rates of electron transport were dependent upon the structure of the polar head group and the length of the hydrocarbon tail. Of the detergents tested, the alkyl maltosides were the best in terms of both high rates of electron transport and superior enzyme stability. With the maltosides, changing the length of the alkyl tail affected the activity of cytochrome c oxidase in a manner quite similar to that reported with synthetic phosphatidylcholines and phosphatidylethanolamines [Vik, S. B., & Capaldi, R. A. (1977) Biochemistry 16, 5755-5759], suggesting that the alkyl maltosides can mimic some of the features of the membrane environment. In each of the detergents, the activation enthalpy (determined from the slope of an Arrhenius plot) was nearly identical, suggesting that the same electron-transfer step within cytochrome c oxidase is rate limiting. This result has been interpreted as evidence for the existence of two or more conformers of cytochrome c oxidase, one of which is significantly more active than the other(s). The enzyme turnover number, which changes by 2 orders of magnitude depending upon the structure of the bound detergent, may reflect the ability of each detergent to alter the equilibrium between the active and nearly inactive conformers.     

Membrane lipids and cytosol carbohydrates in <Emphasis Type="Italic">Aspergillus niger</Emphasis> under osmotic,oxidative, and cold impact

The composition of the membrane lipids and cytosol soluble carbohydrates under three kinds of unfavorable impacts (osmotic, oxidative, and cold) was studied. Changes in the composition of the membrane lipids, specifically, increasing content of phosphatidic acids and decreasing levels of phosphatidylcholines and phosphatidylethanolamines, were the general response to the impacts. The degree of fatty acid unsaturation increased in all dominant phospholipids under osmotic shock, only in cardiolipins and phosphatidic acids under oxidative stress, and only in phosphatidylcholines under cold shock. Increased sterol content was observed only under cold and osmotic treatments. No general pattern was revealed in the composition of cytosol carbohydrates in response to stresses. Oxidative stress had almost no effect on the carbohydrate composition, while osmotic and cold treatments resulted in increased glycerol content and decreased total carbohydrate content. The mechanisms of fungal response to various stress impacts are discussed.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Phospholipase A(2) digestion of cardiolipin bound to bovine cytochrome c oxidase alters both activity and quaternary structure.

Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.     

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that     

Phospholipase digestion of bound cardiolipin reversibly inactivates bovine cytochrome bc1.

Phospholipids and tightly bound cardiolipin (CL) can be removed from Tween 20 solubilized bovine cytochrome bc(1) (EC 1.10.2.2) by digestion with Crotalus atrox phospholipase A(2). The resulting CL-free enzyme exhibits all the spectral properties of native cytochrome bc(1), but is completely inactive. Full electron transfer activity is restored by exogenous cardiolipin added in the presence of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE), but not by cardiolipin alone or by mixtures of phospholipids lacking cardiolipin. Acidic, nonmitochondrial phospholipids, e.g., monolysocardiolipin or phosphatidylglycerol, partially reactivate CL-free cytochrome bc(1) if they are added together with DOPC and DOPE. Phospholipid removal from the Tween 20 solubilized enzyme, including the tightly bound cardiolipin, does not perturb the environment of either cytochrome b(562) or b(566), nor does it cause the autoreduction of cytochrome c(1). Cardiolipin-free cytochrome bc(1) also binds antimycin and myxothiazol normally with the expected red shifts in b(562) and b(566), respectively. However, the CL-free enzyme is much less stable than the lipid-rich preparation, i.e., (1) many chromatographic methods perturb both cytochrome b(566)() (manifested by a hypsochromic effect, i.e., blue shift of 1.5-1.7 nm) and cytochrome c(1) (evidenced by autoreduction in the absence of reducing agents); (2) affinity chromatographic purification of the enzyme causes pronounced loss of subunits VII and XI (65-80% decrease) and less significant loss of subunits I, IV, V, and X (20-30% decrease); and (3) high detergent-to-protein ratios result in disassembly of the complex. We conclude that the major role of the phospholipids surrounding cytochrome bc(1), especially cardiolipin, is to stabilize the quaternary structure. In addition, bound cardiolipin has an additional functional role in that it is essential for enzyme activity.     

Effect of hydrazine on properties of cytochrome oxidase

The effects of hydrazine on ferrocytochrome c oxidation by cytochrome oxidase and on spectral properties of the enzyme were studied. Hydrazine was found to modify the spectral properties of lipid-depleted preparations of cytochrome oxidase dissolved in 1% cholate and to inhibit the cytochrome c oxidase activity of the enzyme, whereas the kinetic properties of lipid-enriched and Tween preparations were unchanged by hydrazine. Cytochrome oxidase was found to possess a hydrazine oxidase activity. This activity was not coupled with the specific cytochrome c oxidase activity. The effect of pH on the observed changes was studied. Hydrazine was found to yield protein bands in the optical spectra of cytochrome oxidase as 580 nm, 537 nm and 845 nm. It is concluded that hydrazine interacts with the oxygen-binding site of cytochrome oxidase. The effect of hydrazine on the formation of the "ferryl" form (Fe4+a3/Cu2+b) of the enzyme is discussed.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Ionic-strength-dependence of the oxidation of native and pyridoxal 5''-phosphate-modified cytochromes c by cytochrome c oxidase.

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.     

Interaction of cytochrome c and its precursor apocytochrome c with various phospholipids

The effects of cytochrome c and apocytochrome c on the structural properties of various membrane phospholipids in model systems were compared by binding, calorimetric, permeability, ³¹P n.m.r. and freeze-fracture experiments. Both cytochrome c and apocytochrome c experience strong interactions only with negatively charged phospholipids; apocytochrome c interacted more strongly than cytochrome c. These interactions are primarily electrostatic but also have a hydrophobic character. Cytochrome c as well as apocytochrome c induces changes in the structure of cardiolipin liposomes as is shown by ³¹P n.m.r. and freeze-fracture electron microscopy. Cytochrome c does not affect the bilayer structure of phosphatidylserine. In contrast, interaction of apocytochrome c with this phospholipid results in changes of the ³¹P n.m.r. bilayer spectrum of the liposomes and also particles are observed at the fracture faces. The results are discussed in relation to the import of the protein into the mitochondrion.     

Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry

The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.