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.     

Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria.

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics. In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147-4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.     

Fusion of phospholipid vesicles mediated by cytochrome c

Fusion of phosphatidylserine/phosphatidylethanolamine (1/1) vesicles induced by cytochrome c is studied at a wide range of pH values. A pH profile for the fusion with maximum values at pH 5 and pH 8 is obtained and this is found to be similar to the profile for cytochrome c binding to the vesicles. The binding property of apocytochrome c to the same phospholipid vesicles is found to be about the same as that of the cytochrome c at low ionic strength, but very different at high salt concentrations. No appreciable fusion of vesicles by apocytochrome c is observed. Proteolytic treatment and dansyl chloride labeling of cytochrome c- and apocytochrome c-vesicle complexes show that the C-terminal segments of these proteins with molecular weights of about 3000 and 5000, respectively, penetrate the bilayer. The hydrophobic labeling studies with photoreactive phosphatidylcholine in the bilayer show that segments of both cytochrome c and apocytochrome c go deep into the bilayer.     

Import of cytochrome c into mitochondria. Cytochrome c heme lyase

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5--10-fold by NADH greater than NADPH greater than glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.     

Membrane-bound and water-soluble cytochrome c1 from Neurospora mitochondria

Cytochrome c1 is a subunit of ubiquinol--cytochrome c reductase (EC 1.10.2.2). In Neurospora crassa wild type 74A grown in the presence of chloramphenicol, the subunit is inserted only into the bilayer of the mitochondrial inner membranes without associating with other proteins. From these modified membranes a monodisperse (cytochrome c1)-Triton complex was isolated by subjecting the Triton-solubilized membranes to affinity chromatography on immobilized cytochrome c. A water-soluble pentamer of cytochrome c1 was prepared from the (cytochrome c1)-Triton complex by removing the detergent. By limited proteolytic digestion of the cytochrome c1-Triton complex with chymotrypsin, a water-soluble monomeric cytochrome c1 was prepared which has a molecular weight of only 24 000 as compared to 31 000 of the membrane-bound cytochrome c1. The 24 000-Mr cytochrome c1 and the 31 000-Mr cytochrome c1 have same light absorption spectra and cytochrome-c-binding properties. These results are used to propose the following model. Cytochrome c1 consists of a large hydrophilic part and a small hydrophobic part. The hydrophilic part extends from the mitochondrial inner membrane into the intermembrane space. This part carries the heme and interacts with cytochrome c. The hydrophobic part anchors the cytochrome c1 to the bilayer.     

Intramembranous particles and ripples in lipid-cytochrome c bilayers

Vesicles made from a mixture of phospholipids (PL) are unilamellar. When such lipid vesicles are incubated with reduced cytochrome c, multilamellar vesicles (PLC) are visualized by transmission electron microscopy. Both PL and PLC vesicles show a rippling in the freeze-fracture replicas. The rippling effect is likely to be due to the tilting of fatty acid chains. The freeze-fractured faces of PLC vesicles also show 3–4 nm intramembranous particles. Such particles are lacking from the comparable fractured faces of PL vesicles that are produced in the absence of cytochrome c. It is suggested that these intramembranous particles result from the presence of cytochrome c molecules; therefore, cytochrome c molecules may penetrate the lipid bilayer.     

Interaction of an anticancer ruthenium complex HInd[RuInd2Cl4] with cytochrome c

Cytochrome c is an important electron transfer protein in the respiratory chain, shuttling electrons from cytochrome c reductase to cytochrome c oxidase. Extensive chemical modification studies indicate significant electrostatic interactions between these proteins and show that all structural and conformational changes of cytochrome c can influence the electron transport. In the present work we examine the effect of an anticancer ruthenium complex, trans-Indazolium (bisindazole) tetrachlororuthenate(III) (HInd[RuInd(2)Cl(4)]), on the conformation of cytochrome c, the state of the heme moiety, formation of the protein dimer and on the folding state of apocytochrome c. For this purpose, gel-filtration chromatography, absorption second derivative spectroscopy, circular dichroism (CD) and inductively coupled plasma atomic emission spectroscopy (ICP(AES)) were used. The present data have revealed that binding of the potential anticancer drug HInd[RuInd(2)Cl(4)] complex to cytochrome c induces a conformation of the protein with less organized secondary and tertiary structure.     

Investigations on the insertion of the mitochondrial precursor protein apocytochrome c into model membranes

Different aspects of the interaction of apocytochrome c and model membranes composed of negatively charged lipids, were studied in order to get insight into the nature of this interaction. The effect of the protein on the lipid packing properties are revealed by DSC, ESR and monolayer techniques. These experiments clearly demonstrate that upon electrostatic interaction with the negatively charged phospholipids, apocytochrome c is able to penetrate into the hydrophobic region of the model membrane. In the case of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, this results in a perturbation of 160 lipid molecules per apocytochrome c molecule. Most likely, apocytochrome c disrupts the formation of the gel phase and restricts the lipid chain motion above the gel to liquid-crystalline phase transition. Tryptophan fluorescence measurements confirm that at least a part of the protein penetrates into the bilayer, and suggest that after this penetration, the tryptophan (residue no. 59) is located in the glycerol backbone region of the phospholipids. Although the secondary structure of apocytochrome c is predicted to contain about 35% of alpha-helical structure, the CD pattern of an aqueous solution of the protein is featureless. However, negatively charged lipids are able to express this alpha-helical potency in the apocytochrome c, which might be important for the insertion of the protein into lipid membranes.     

Evidence for formation of two thioether bonds to link heme to apocytochrome c by partially purified cytochrome c synthetase

Cytochrome c synthetase has been solubilized from yeast mitochondria using Triton X-100 and fractionated with ammonium sulfate. Use of this partially purified enzyme has permitted us to isolate a quantity of iso-1-cytochrome c formed from 125I-labeled apocytochrome c and hemin in the presence of a NADPH-generating system. Visible absorption spectra (pH 8.0 or 5.0) including alpha, beta, and Soret bands and their molar absorption coefficients of this enzymatically synthesized cytochrome c in the oxidized and reduced states are the same, within experimental error, as those of native cytochrome c. Pyridine ferrohemochrome (pH 13) of the synthesized species also exhibits the same alpha and beta bands as those of iso-l-cytochrome c and similar to those reported for heme peptides of cytochrome c. If only one or no thioether bond were formed between the two vinyl side groups of heme and the cysteine residues of apocytochrome c, all these alpha and beta bands would have shifted to red (Pettigrew, G. W., Leaver, J. L., Meyer, T. E., and Ryle, T. E. (1975) Biochem J. 147, 291-302). Thus, two thioether bonds appear to be formed to link heme to apocytochrome c by cytochrome c synthetase, completing information of the three-dimensional structure of cytochrome c.     

Apocytochrome c interaction with phospholipid membranes studied by Fourier-transform infrared spectroscopy.

Apocytochrome c, the heme-free precursor of cytochrome c, has been used extensively as a model to study molecular aspects of posttranslational translocation of proteins across membranes. In this report, we have used Fourier-transform infrared spectroscopy to gain further insight into the mechanism of apocytochrome c interaction with membrane phospholipids. Association of apocytochrome c with model membranes containing the acidic lipid dimyristoylphosphatidylglycerol (DMPG) as a single component results in a drastic perturbation of phospholipid structure, at the level of both the acyl chains and the interfacial carbonyl groups. However, in a binary mixture of DMPG with acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54), the perturbing effect of the protein on the acidic phospholipid is greatly attenuated. In such a membrane with mixed lipids, the physical properties of the DMPG and DMPC components are affected in a similar fashion, indicating that apocytochrome c does not induce any significant segregation or lateral-phase separation of acidic and zwitterionic lipids. Analysis of the apocytochrome c spectrum in the amide I region reveals that binding to phospholipids causes considerable changes in the secondary structure of the protein, the final conformation of which depends on the lipid to protein ratio. In the presence of a large excess of DMPG, apocytochrome c undergoes a transition from an essentially unordered conformation in solution to an alpha-helical structure. However, in complexes of lower lipid to protein ratios (less than or equal to approximately 40:1), infrared spectra are indicative of an extended, intermolecularly hydrogen-bonded beta-sheet structure. The latter is suggestive of an extensive aggregation of the membrane-associated protein.     

Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures: Structural changes between hexagonal,cubic and bilayer phases

The polymorphic phase behaviour of dilinoleoylphosphatidyethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is investigated by freeze-fracture electron microscopy, X-ray diffraction and ³¹P-NMR. The structures at 5% or less POPC are predominantly inverted hexagonal (H_II), whereas at 15% or more POPC, the structure is mostly bilayer (L), interrupted by defects (lipidic particles). A cubic phase structure is observed in the transition range between H and L phases; the cubic arrangement deteriorates at higher temperatures into an amorphous aggregate of spherical units. Both cubic and amorphous structures contribute to the isotropic ³¹P resonance, with no preference for PC or PE partitioning in the isotropic motion as observed by high resolution NMR. The existence of the cubic phase seems to depend critially on the homogeneity and the degree unsaturation of the phospholipids.     

Structural preferences of phosphatidylinositol and phosphatidylinositol-phosphatidylethanolamine model membranes influence of Ca2+ and Mg2+

The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca²⁺ and Mg²⁺ on these preferences, have been examined employing ³¹P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca²⁺ and Mg²⁺. In mixed systems with H_II phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (<10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca²⁺ (but not Mg²⁺) can induce H_II phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca²⁺ induces structural reorganization in mixed systems containing acidic phospholipids and phosphatidylethanolamine.     

未培养厌氧甲烷氧化古菌来源细胞色素c的异源表达优化

细胞色素c是一类在生物体内广泛存在的血红素蛋白,由亚铁血红素和细胞色素c前体组成,在生物电子学、生物医药以及污染物降解等领域具有很大的潜在应用价值.然而,细胞色素c很难通过异源表达而大量获取.对于未培养厌氧甲烷氧化古菌来源的细胞色素c (CytC4),目前尚无成功表达和功能研究.本研究首先通过在CytC4的N端分别引入...     

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.     

Differential interactions of apo- and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria

Monomolecular layers of lipid extracts of microsomal, mitochondrial outer and inner membranes, and pure lipid species have been used to measure their interaction with apo- and holocytochrome c. Large differences were observed both with respect to the nature and the lipid specificity of the interaction. The initial electrostatic interaction of the hemefree precursor apocytochrome c with anionic phospholipids is followed by penetration of the protein in between the acyl chains. Apocytochrome c shows similar interactions for all anionic lipids tested. In strong contrast the holoprotein discriminates enormously between cardiolipin for which it has a high affinity and phosphatidylserine and phosphatidylinositol for which it has a much lower affinity. For these latter lipids the interaction with cytochrome c is primarily electrostatic. The cytochrome c-cardiolipin interaction shows several unique features which suggest the formation of a specific complex between the two molecules. These properties account for the preference in interaction of the apoprotein with the lipid extract of the outer mitochondrial membrane over that of the endoplasmic reticulum and the large preference of cytochrome c for the inner over that of the outer mitochondrial membrane lipid extract. Only apocytochrome c was able to induce close contacts between monolayers of the mitochondrial outer membrane lipids and vesicles of mitochondrial inner membrane lipids. Experiments with fragments of both protein and unfolding experiments with cytochrome c revealed that the differences in interaction between the two proteins are mainly due to differences in their tertiary structure and not the presence of the heme group itself. The initial unfolded structure of apocytochrome c is responsible for the high penetrative power of the protein and its ability to induce close membrane contact, whereas the folded structure of cytochrome c is responsible for the specific interaction with cardiolipin. The results are discussed in the light of the apocytochrome c import process in mitochondria and suggest that lipid-protein interactions contribute to targeting the precursor toward mitochondria and are important for its translocation across the outer mitochondrial membrane and the final localization of cytochrome c toward the outside of the inner mitochondrial membrane.     

Characterization of the reaction products of cytochrome c with glutathione by mass spectrometry

Cytochrome c and glutathione (GSH) are two important biomolecules that regulate many cellular processes. The reaction of cytochrome c with GSH involves radical oxygen species and exhibits significant complexity. In the present work, the reaction of cytochrome c with GSH in water was characterized using mass spectrometry. The results show for the first time that the reaction generates multiple products including apocytochrome c in oxidized and reduced forms, glutathionylated apocytochrome c, GSH-modified cytochrome c, and oxidized and hydroxylated species. The reaction is O(2) dependent and is rapid in water at neutral pH and 37 degrees C. The reaction involves the cleavage of thioether linkages between the heme and apocytochrome c. Evidence for the role of H(2)O(2) and other oxygen radicals in this reaction is also provided.     

Effects of adriamycin on lipid polymorphism in cardiolipin-containing model and mitochondrial membranes

(1) The effects of the anti-tumor drug adriamycin on lipid polymorphism in cardiolipin-containing model membranes and in isolated inner mitochondrial membranes has been examined by ³¹P-NMR. (2) Adriamycin binding does not affect the macroscopic structure or local order in the phosphate region of cardiolipin liposomes. (3) In cardiolipin liposomes and in cardiolipin-phosphatidylcholine (1:1) liposomes, the drug inhibits the ability of Ca²⁺ to induce the hexagonal H_II phase. (4) Adriamycin interaction with both dioleoylphosphatidylethanolamine-cardiolipin (2:1) and dioleoylphosphatidylethanolamine-phosphatidylserine (1:1) liposomes results in structural phase separation into a liquid-crystalline hexagonal H_II phase for the phosphatidylethanolamine and a liquid-crystalline lamellar phase for the negatively charged phospholipid. (5) Combined high-resolution ³¹P-NMR, electron microscopy and light scattering studies reveal the prominent fusion capacity of adriamycin towards cardiolipin-phosphatidylcholine small unilamellar vesicles. (6) Addition of Ca²⁺ to total rat liver inner mitochondrial membrane lipids, dispersed in excess buffer, results in hexagonal H_II formation for part of the phospholipids. By contrast, the original bilayer structure is completely conserved when the above experiment is performed in the presence of adriamycin. (7) ³¹P-NMR spectra of isolated inner mitochondrial membranes are indicative of a bilayer organization for the majority of the phospholipids. Approximately 15% of the signal intensity originates from phospholipids which experience isotropic motion. Adriamycin addition almost completely eliminates the latter spectral component. In the absence of adriamycin, Ca²⁺ addition greatly increases the percentage of the phospholipids giving rise to an isotropic signal possibly indicating the formation of non-lamellar lipid structures. Adriamycin which specifically binds to cardiolipin (K. Nicolay et al. (1984) Biochim. Biophys. Acta 778, 359–371) completely blocks the Ca²⁺-induced structural reorganization of the lipids in this membrane.     

Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.