Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase
2007; Springer Nature; Volume: 26; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7601618
ISSN1460-2075
AutoresKyoko Shinzawa‐Itoh, Hiroshi Aoyama, Kazumasa Muramoto, H. Terada, T. Kurauchi, Y. Tadehara, Akiko Yamasaki, Takashi Sügimura, Sadamu Kurono, Kazuo Tsujimoto, Tsunehiro Mizushima, Eiki Yamashita, Tomitake Tsukihara, Shinya Yoshikawa,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle1 March 2007free access Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase Kyoko Shinzawa-Itoh Kyoko Shinzawa-Itoh Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Hiroshi Aoyama Hiroshi Aoyama RIKEN Harima Institute, Mikazuki Sayo, Hyogo, Japan Search for more papers by this author Kazumasa Muramoto Kazumasa Muramoto Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Hirohito Terada Hirohito Terada Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Tsuyoshi Kurauchi Tsuyoshi Kurauchi Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Yoshiki Tadehara Yoshiki Tadehara Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Akiko Yamasaki Akiko Yamasaki Department of Material Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Takashi Sugimura Takashi Sugimura Department of Material Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Sadamu Kurono Sadamu Kurono Graduate School of Material Science, Japan Advanced Institute of Science and Technology, Nomi Ishikawa, Japan Search for more papers by this author Kazuo Tsujimoto Kazuo Tsujimoto Graduate School of Material Science, Japan Advanced Institute of Science and Technology, Nomi Ishikawa, Japan Search for more papers by this author Tsunehiro Mizushima Tsunehiro Mizushima Institute for Protein Research, Osaka University, Japan Search for more papers by this author Eiki Yamashita Eiki Yamashita Institute for Protein Research, Osaka University, Japan Search for more papers by this author Tomitake Tsukihara Tomitake Tsukihara Institute for Protein Research, Osaka University, Japan Search for more papers by this author Shinya Yoshikawa Corresponding Author Shinya Yoshikawa Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Kyoko Shinzawa-Itoh Kyoko Shinzawa-Itoh Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Hiroshi Aoyama Hiroshi Aoyama RIKEN Harima Institute, Mikazuki Sayo, Hyogo, Japan Search for more papers by this author Kazumasa Muramoto Kazumasa Muramoto Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Hirohito Terada Hirohito Terada Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Tsuyoshi Kurauchi Tsuyoshi Kurauchi Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Yoshiki Tadehara Yoshiki Tadehara Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Akiko Yamasaki Akiko Yamasaki Department of Material Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Takashi Sugimura Takashi Sugimura Department of Material Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Sadamu Kurono Sadamu Kurono Graduate School of Material Science, Japan Advanced Institute of Science and Technology, Nomi Ishikawa, Japan Search for more papers by this author Kazuo Tsujimoto Kazuo Tsujimoto Graduate School of Material Science, Japan Advanced Institute of Science and Technology, Nomi Ishikawa, Japan Search for more papers by this author Tsunehiro Mizushima Tsunehiro Mizushima Institute for Protein Research, Osaka University, Japan Search for more papers by this author Eiki Yamashita Eiki Yamashita Institute for Protein Research, Osaka University, Japan Search for more papers by this author Tomitake Tsukihara Tomitake Tsukihara Institute for Protein Research, Osaka University, Japan Search for more papers by this author Shinya Yoshikawa Corresponding Author Shinya Yoshikawa Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan Search for more papers by this author Author Information Kyoko Shinzawa-Itoh1,‡, Hiroshi Aoyama2,‡, Kazumasa Muramoto1, Hirohito Terada1, Tsuyoshi Kurauchi1, Yoshiki Tadehara1, Akiko Yamasaki3, Takashi Sugimura3, Sadamu Kurono4, Kazuo Tsujimoto4, Tsunehiro Mizushima5, Eiki Yamashita5, Tomitake Tsukihara5 and Shinya Yoshikawa 1 1Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan 2RIKEN Harima Institute, Mikazuki Sayo, Hyogo, Japan 3Department of Material Science, University of Hyogo, Kamigohri Akoh Hyogo, Japan 4Graduate School of Material Science, Japan Advanced Institute of Science and Technology, Nomi Ishikawa, Japan 5Institute for Protein Research, Osaka University, Japan ‡These authors contributed equally to this work *Corresponding author. Department of Life Science, University of Hyogo, Kamigohri Akoh Hyogo 678-1297, Japan. Tel.: +81 791 58 0190; Fax: +81 791 58 0132; E-mail: [email protected] The EMBO Journal (2007)26:1713-1725https://doi.org/10.1038/sj.emboj.7601618 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info All 13 lipids, including two cardiolipins, one phosphatidylcholine, three phosphatidylethanolamines, four phosphatidylglycerols and three triglycerides, were identified in a crystalline bovine heart cytochrome c oxidase (CcO) preparation. The chain lengths and unsaturated bond positions of the fatty acid moieties determined by mass spectrometry suggest that each lipid head group identifies its specific binding site within CcOs. The X-ray structure demonstrates that the flexibility of the fatty acid tails facilitates their effective space-filling functions and that the four phospholipids stabilize the CcO dimer. Binding of dicyclohexylcarbodiimide to the O2 transfer pathway of CcO causes two palmitate tails of phosphatidylglycerols to block the pathway, suggesting that the palmitates control the O2 transfer process.The phosphatidylglycerol with vaccenate (cis-Δ11-octadecenoate) was found in CcOs of bovine and Paracoccus denitrificans, the ancestor of mitochondrion, indicating that the vaccenate is conserved in bovine CcO in spite of the abundance of oleate (cis-Δ9-octadecenoate). The X-ray structure indicates that the protein moiety selects cis-vaccenate near the O2 transfer pathway against trans-vaccenate. These results suggest that vaccenate plays a critical role in the O2 transfer mechanism. Introduction Integral membrane proteins contain various specifically bound lipids, whose hydrophobic tails provide non-polar and highly anisotropic environments in the protein interior. These anisotropic environments, which cannot be created without lipids, are expected to provide unique protein functions that occur only within the interior of the protein. Extensive characterization of integral lipids in the context of complete structural characterization of a given membrane protein is necessary for elucidation of the mechanism of the physiological function of membrane proteins. Generally speaking, the resolution of X-ray structures of various membrane proteins reported thus far (Marsh and Pali, 2006) is not sufficiently high for determination of the lipid structures, particularly the unsaturated bond positions and the chain lengths of the hydrophobic tails. Mass spectrometry (MS) and tandem mass spectrometry (MS-MS) are powerful tools for analysis of the hydrophobic tail structures. Phospholipase treatment is required for determination of the position of each acyl group in the glycerol backbone. However, complete lipid structural determinations have been reported only for lipids of bacteriorhodopsin and the purple membrane (Corcelli et al, 2000). The lipids identified in the X-ray structure of bacteriorhodopsin surround the protein in a manner similar to that of the purple membrane (Belrhali et al, 1999), suggesting that the lipid–protein interactions are nonspecific under the physiological conditions. On the other hand, most of the phospholipids found in X-ray structures of the other membrane proteins are tightly bound as structural elements or prosthetic groups. In fact, a cardiolipin (CL), a phosphatidylcholine (PC) and a glucosylgalactosyl diacylglycerol have been identified in the 2.1 and 2.55 Å X-ray structures of bacterial reaction centers, which indicate the existence of various specific lipid/protein interactions (McAuley et al, 1999; Camara-Artigas et al, 2002). Five phospholipid molecules identified in yeast cytochrome bc1 complex at 2.3 Å resolution appear to play specific roles in facilitating structural and functional integrity of the enzyme. Direct involvement of a CL in the bc1 complex for the proton uptake has been proposed (Lange et al, 2001). Several phospholipids have been identified in the X-ray structures of bovine and bacterial cytochrome c oxidase (CcO) but chemical structural confirmation of the assignments has not been provided (Tsukihara et al, 1996; Harrenga and Michel, 1999; Svensson-Ek et al, 2002). CL binding to CcO has been examined by photolabeling (Sedlák et al, 2006). Here, we report X-ray structures of all 13 lipids contained in a bovine heart CcO preparation, stabilized with decylmaltoside, and chemical structures of these lipids including the chain lengths and the positions of the unsaturated bonds of the hydrophobic tails. Chemical structures of lipids contained in the mitochondrial inner membrane, the other mitochondrial proton-pumping respiratory complexes and the isolated CcO of Paracoccus denitrificans, the ancestor of eukaryotic mitochondrion, have been analyzed by MS spectrometry, for evaluation of the physiological significance of these lipid structures in CcO. Results Chemical structural analyses of integral lipids of bovine heart CcO Phospholipids. Mass spectra of the lipid fraction extracted from CcO purified with crystallization as the final step (hereafter referred to as crystalline CcO) were obtained with an electrospray ionization-time of flight (ESI-TOF) mass spectrometer (Accu TOF, JEOL, JMS-T100LC). The spectra showed six peaks assignable to phospholipids at mass/ion values (m/z) of 742.58, 764.56, 766.58, 780.55, 782.57 and 790.54 in the positive ion mode and six peaks at m/z values of 723.50, 747.52, 766.54, 1446.81, 1447.89 and 1448.97 in the negative ion mode (Figure 1). (In the positive ion mode, Na+ adducts provide these peaks. Thus, the m/z values are 22.99 higher than the true molecular mass values. The values are 1.01 lower in the negative ion modes owing to deprotonation.) Figure 1.Mass spectra of lipid fraction extracted from the crystalline bovine heart CcO preparation, using an Accu TOF (JEOL, JMS-T100LC) mass spectrometer with methanol. The upper and lower spectra are those in the positive and negative ion modes, respectively. Only the m/z regions where phospholipid signals as shown by arrows are observable are given. Download figure Download PowerPoint The spectra show many minor bands owing to naturally abundant isotope species, which sometimes overlap with peaks arising from different chemical species having similar molecular weights. For example, the 766.58 peak is near the position of one of the isotope species of the 764.56 species. However, the position of the 766.58 peak is distinguishable from the isotope responsible for the 766.56 peak, as the machine accuracy of the m/z value is less than 5 p.p.m. Furthermore, the 766.58 peak clearly has a higher intensity than that expected for the isotope of the 764.56 species (about 10% of the 764.56 peak intensity). Thus, the 766.58 peak is obviously distinct from the isotope of the 764.56 species. The species showing the 764.56 peak as the Na+ adduct is responsible for the 742.58 peak as the H+ adduct instead of the Na+ adduct. The peak near 767.5 m/z (marked by X) is likely to be due to a trace of a contaminant species as no other reasonable assignment is possible. The six smaller m/z values in the positive ion mode coincide with the molecular weights of two types of choline plasmalogens (CP) (C42H80NO7P (C=16:0, 18:2) and C42H82NO7P (C=16:0, 18:1)) and PC (C42H80NO8P (C=16:0, 18:2) and C42H82NO8P (C=16:0, 18:1)). The carbon atoms of the glycerol backbone are numbered as follows: C-1 represents the carbon atom at the opposite terminus of the phosphate-bearing carbon; C-2 represents the middle carbon atom; and C-3 represents the phosphate-bearing carbon atom. The numbers of carbon atoms and unsaturated bonds in the C-1 position (W and X) and those in the C-2 position (Y and Z) are denoted by C=W:X, Y:Z in this paper. Using this description, the saturated fatty acyl groups are provisionally placed at the C-1 position. The predictions for the fatty acyl group position were confirmed by the phospholipase A2 treatment as described below. The 790.54 peak indicates the presence of a phosphotidylethanolamine (PE) (C=18:0, 20:4) carrying one sodium ion. The 766.54 peak in the negative ion mode shows the molecular weight of PE (C=18:0, 20:4). The 747.52 peak position coincides with the molecular weight of a phosphatidylglycerol (PG) (C=16:0, 18:1). The 1448.97 peak coincides with that of isolated bovine heart CL (C=18:2 in all the four positions). The 1447.89 peak is assignable to a CL complexed to a decylmaltoside trimer, which has two negative charges. Decylmaltoside was employed as a detergent for stabilization of CcO in aqueous solution. The 1446.81 peak is assignable to the decylmaltoside trimer. The 723.50 m/z value is that of a dianion of CL (1448.97) generated by removal of two protons. The same complexes and the dianion were also observed in a measurement of a mixture of the authentic chemicals. The assignments of peaks of PG and the other phospholipids were confirmed by MS analyses carried out under the same conditions for a synthetic PG (1-palmitoyl-2-oleylphosphatidylglycerol) and these commercially available phospholipids isolated from bovine heart, respectively (data not shown). For further structural analyses, the lipid extract obtained from the crystalline CcO was fractionated by a two-dimensional thin-layer chromatography (2D-TLC) into four components, CL, PE, PG and a CP/PC mixture (experimental details are provided in Supplementary data 1). MS-MS analysis of each phospholipid fraction confirmed the fatty acid composition as follows: CL contains only a single fatty acid, which has an m/z value of 279. Two types of fatty acids with m/z values of 283 and 303 were observed for PE. PG contained two fatty acid species with m/z values of 255 and 281. The two PC species with m/z values of 742 and 744 provided two fatty acid pairs, (255, 279) and (255, 281), respectively. The structures of each of the fatty acids were determined by MS–MS analyses of the fatty acid fraction isolated by TLC from a phospholipase lysate of each phospholipid, as described in Materials and methods. The positions of each fatty acid in the glycerol backbone were determined after limited treatment with phospholipase A2 under conditions such that only the ester at the C-2 position is cleaved. Structures of the saturated fatty acids were determined by MS-MS spectral analyses. The MS-MS spectra of these fatty acids with unsaturated bonds were compared with those of the authentic commercially available samples to identify the positions of the unsaturated bonds. By these analyses, the structures of the fatty acids were determined as follows: the fatty acid with the m/z value of 279 arises from CL (CL279): linoleate (Δ9,12-octadecadienoate), PE283: stearate (n-octadecanoate), PE303:arachidonate (Δ5,8,11,14-eicosatetraenoate), PG255: palmitate (n-hexadecanoate), PG281: vaccenate (Δ11-octadecenoate), PC255: palmitate, PC279: linoleate and PC281: oleate (Δ9-octadecenoate) (experimental details for the MS-MS analyses are given in Supplementary data 2). These MS-MS analyses, together with the phospholipase treatment results, indicate that PC, PE and PG contain saturated and unsaturated fatty acyl groups at the C-1 and C-2 positions, respectively, and that CL contains only the linoleic acyl group at all four positions. The phospholipase A2 treatment was performed for the CP/PC mixture without separation, to provide three types of fatty acids, with m/z values of 279 and 281 for the C-2 position and 255 for the C-1 position, consistent with the results of MS-MS analyses of the two PC species. The differences in both of the m/z values between the two PCs and between the two CPs are 2.02, whereas those between the larger PC and CP and between the smaller PC and CP are 16.01, as shown in Figure 1. These results strongly suggest that the structure of each CP is identical to that of the derivative of the corresponding PC. The derivative has a vinylic ether instead of an ester. The configuration of vaccenate in PG was determined by GC analysis of the methyl ester obtained by solvolysis of lipid fractions of bovine heart CcO (Supplementary data 3-1). The peak positions of methyl esters of cis-oleate, cis-vaccenate and trans-vaccenate are separated well enough for quantitative analysis of the peak areas. The GC analyses indicate that the cis/trans ratio is approximately 12.8:1. The trans isomer was not detected for the oleate from PC. The cis/trans configuration was not examined for the linoleate and arachidonate molecules detected in the crystalline enzyme (experimental details are in Supplementary data 3-1). The chemical structures of the seven species of phospholipids detected in the crystalline bovine heart CcO are given in Figure 2. The cis-configuration of the unsaturated bonds shown in the figure except for that of vaccenate and oleate is provisional. Figure 2.Chemical structures of phospholipids detected in crystalline bovine heart CcO. The major configuration (cis) for vaccenate and oleate is shown. The cis-configuration is provisionally assigned for the other unsaturated fatty acids. Download figure Download PowerPoint Triglycerides. An extremely hydrophobic spot was detected in the 2D-TLC pattern (Supplementary data 1). Phosphorous and sugar were not detected in the spot. An extract of the spot gave a proton NMR spectrum, which was superimposable with that of trioleil glycerol, indicating that the spot obtained in the 2D-TLC is a mixture of triglycerides. The MS spectrum of the fraction in the ESI positive ion mode showed seven peaks at m/z 855.74, 879.74, 881.75, 885.79, 905.76, 907.77 and 909.79 (NMR and MS spectra are given in Supplementary data 4). The fatty acid structures and compositions were examined by GC analysis as the methyl esters obtained by solvolysis. Four species of fatty acid methyl esters were identified, as follows: palmitate (C=16:0), stearate (C=18:0), oleate (C=18:1) and linoleate (C=18:2). The fatty acid compositions of the seven triglycerides (TGs) were estimated by the molecular weights of these TGs, as given in Table I. The molar fractions of each of these four methyl esters were determined by a quantitative GC analysis to be 17% palmitate, 12% stearate, 61% oleate and 10% linoleate (Supplementary data 3-2). The fatty acid positions within the glycerol backbone were not determined. Table 1. Fatty acid compositions of the seven TGs in bovine heart CcO Triacyl glycerol m/z Fatty acid composition Palmitate Stearate Oleate Linoleate TG1 855.74 2 1 TG2 879.74 1 1 1 TG3 881.75 1 2 1 1 1 TG4 885.79 1 2 TG5 905.76 2 1 1 2 TG6 907.77 3 1 1 1 TG7 909.79 1 2 2 1 Palmitate: n-hexadecanoate; stearate: n-octadecanoate; oleate: Δ9-octadecenoate; linoleate: Δ9, 12-octadecadienoate. A possible structure of TG6: The fatty acid compositions of TG in the bovine heart adipose tissue were also examined and were found to be identical to those of the TG fraction obtained from the crystalline bovine heart CcO. Thus, we examined whether these TGs were contaminated during the purification procedure for the enzyme from the bovine heart muscle by addition of significant amount of unnatural TG (containing fatty acyl groups with odd-numbered carbon chains) to the heart muscle mitochondrial fraction before initiation of the purification procedure (Supplementary data 4). The results indicate that TG contamination arising from adipose tissue during the purification procedure was not detected. Quantitative analysis of the total fatty acids of bovine heart CcO. The total amount of fatty acyl groups was estimated by extraction of lipids in the presence of an unnatural TG, tripentadecanoic glyceride (15:0), as an internal standard at the level of 2 molecules of TG per one enzyme molecule followed by the solvolysis with methanol. The solvolysate was quantitatively examined by GC using the conditions and correction factors indicated in Supplementary data 3-2 and 3-3. The six determinations provide 30.6 (±3.4) fatty acids per enzyme molecule. Under the present GC conditions, arachidonate is not detectable. Thus, this value represents the amount of fatty acids other than arachidonate. Phosphorous content of crystalline bovine heart CcO. Phosphorous content was determined for 40 different batches of the crystalline preparation and was found to be 12.9±1.9 phosphorous/mol of the enzyme. Lipid compositions in the mitochondrial inner membrane and other proton-pumping respiratory complexes MS spectral analysis of the bovine heart mitochondrial inner membrane fraction indicated that this fraction contained the same seven species of the phospholipids as those identified in bovine heart CcO, although the relative intensities of the peaks are different (Figure 3A). The phospholipid composition of the bovine heart mitochondrial inner membrane determined from the present MS analyses is essentially consistent with the reported results for the major phospholipids (Daum, 1985). Minor constituents, such as phosphatidylinositol, phosphatidylserine and sphingomyelin, were not detected under the present conditions. The intensity ratio of the two major peaks located in close proximity to each other provides a reliable standard for evaluation of a change in the composition. The other proton-pumping respiratory complexes, NADH–ubiquinone reductase, ubiquinol–cytochrome c reductase and Fo F1ATP synthase (known as complexes I, III and V, respectively), also contain only the same seven species of phospholipids, but with variable intensity ratios (spectra are provided in Supplementary data 5). The intensity ratio of the 747.52 peak (PG) to the 766.54 peak (PE) detectable in the ESI negative mode is extremely high in CcO (6.0), relative to 0.7, 0.5, 0.3 and 0.3 for complexes I, III and V and the mitochondrial inner membrane, respectively. The cis/trans ratio of vaccenate in the bovine heart mitochondrial inner membrane was found to be 5/1 (Supplementary data 3-1), which is notably lower than that of the vaccenate in the bovine heart CcO (12.8/1). TG was detectable in the mitochondrial inner membrane, whereas complexes I, III and V did not contain significant amounts of TG. The specificity suggests that specific binding of TG to CcO occurs. Figure 3.Mass spectra of lipid extracts from the bovine heart mitochondrial inner membrane (A) and the purified CcO (B) of P. denitrificans. These spectra were obtained under the same conditions described for Figure 1. No signal assignable to phospholipid was detected in the other m/z regions. Download figure Download PowerPoint The lipid composition of CcO isolated from the soil bacterium P. denitrificans, the ancestor of eukaryotic mitochondria, was determined in order to evaluate the physiological significance of the lipids in CcO. Only two types of PG (C=16:0, 18:1 and C=18:0, 18:2) were detected (Figure 3B). GC analysis for the solvolysate of PG showed that the fatty acid (18:1) is vaccenate and that no significant amounts of trans-vaccenate were observed (data in Supplementary data 3-1). X-ray structures of lipids in bovine heart CcO Overall X-ray structures of lipids. The structure of lipids determined by the chemical analyses described above fits well within the electron density map of 1.8 Å resolution X-ray structure. The best-fit atomic model (PDB code 2DYR) shows 2CLs, 1PC, 3PEs, 4PGs and 3TGs (Figure 4A and B). The structures determined by the chemical analyses fit well within the (FO−FC) difference electron density map. The structures of lipids were refined to give reasonable fatty acid tail conformations in which the torsion angles of single bonds in the fatty acid tails consistently adopt the energetically favored staggered conformations. Eclipsed conformations of single bonds within the fatty acid tails are not present in the X-ray structure (Figure 4C). All four species of phospholipids containing choline (two types of PC and two types of CP) identified by the chemical analyses fit equally well within the electron density of the single PC site. The seven TG species are also fit equally well within each of the three TG sites. Four cholates and two decylmaltosides are specifically bound, as shown in Figure 4. Figure 4.The locations of lipids and detergents in the X-ray structure of bovine heart CcO at 1.8 Å resolution in the oxidized state in side (A) and top (B) stereo-views. CH and DM denote cholate and decylmaltoside, respectively. Association of lipids and detergents with individual monomers is indicated by rectangles. The numbering system for each lipid and detergent species is arbitrary. The Cα-backbone traces are only given for the protein moiety. A histogram indicating the populations of torsion angles of single bonds of the hydrocarbon tails of all lipids in the X-ray structure is given in (C). Two plateau regions near ±120° are detectable in the population. There are no torsion angles less than ±30°. Download figure Download PowerPoint The X-ray structure of CcO has 33 sites for accepting the hydrophobic tails. This observation is consistent with the quantitative determination of fatty acid tails of 30.6±3.4 under conditions in which the three arachidonate moieties of PE are not detectable. Furthermore, the assignment of the phospholipids to the electron density is fully consistent with the phosphorous content analysis, which indicates the presence of 13 phosphorous atoms including one phosphate of the phosphothreonine as described below and 12 phosphate-derived phosphate head groups. Thus, all lipids detected in the fatty acid tail analyses are identified in the X-ray structure and represent integral lipids that are specifically bound to the protein. As shown in Figure 4, all of the lipids are generally located roughly within the transmembrane helix region on either the positive side or the negative side. The positive and negative sides denote the sides of CcO facing outside and inside of the mitochondrial inner membrane, respectively. The head groups of the phospholipids and the glycerol backbones of TGs are generally located on either of the planes at both ends of the transmembrane helix region. The two planes are about 30 Å apart (Supplementary data 6). The bilayer-like arrangement has also been identified in the bacterial reaction center (Camara-Artigas et al, 2002) and in the yeast cytochrome bc1 complex (Lange et al, 2001). These observations suggest the existence of a common mechanism for lipid incorporation into the membrane proteins. Three phospholipids and one TG are located within the positive side, whereas the remaining lipids are located within the negative side. PG1, PG2 and PE2 are surrounded by the two helix bundles of subunit III. The remaining lipids contribute to stabilization of the dimeric state and the assemblies of nuclear-coded transmembrane subunits with the three core subunits (subunits I, II and III), which are encoded by mitochondrial genes. The locations of these head groups and the interactions between these lipids and the 13 subunits are schematically summarized in Supplementary data 6. Many hydrophobic interactions were detected including typical CH–π interactions (structural details in Supplementary data 7). Lipid–protein interactions Phospholipids in subunit III. The fatty acid tails of the two PGs located within the negative side in subunit III are essentially fully extended, whereas the arachidonyl tail of PE located within the positive side is significantly more folded than the other five fatty acid tails (Figure 5A). The fatty acid tails are fixed tightly by the protein portion, giving clear electron density maps as shown in Figure 5B. The head groups are tightly fixed by many protein-derived hydrogen bonds (Supplementary data 8). The average total numbers of hydrogen bonds and the hydrophobic interactions to the polar and non-polar moieties of the phospholipids are 12.0 and 98.3, respectively, for the three phospholipids in subunit III, and 5.6 and 54.6 for the other phospholipids. The average overall temperature factors for the phospholipids inside and outside subunit III are 40.4 and 83.2 Å2, respectively. The average overall temperature factors for the tail portions inside and outside subunit III are 44.7 and 80.2 Å2, respectively. The average for the head group within subunit III is 26.3 Å2. This value is even lower than that of the surrounding protein portion (Table II). The average for the head groups of phospholipids outside subunit III is 95.3 Å2. As shown in Figure 5A,
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