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Lipid region of the positive-ion reflectron MALDI-ToF spectrum of cytochrome c oxidase sample CcO1 using DHB as the matrix. One microliter of CcO1 enzyme solution at 10 mM was mixed with 1 mL of DHB on the sample target. MGD(monogalactosyldiacylglycerol), PC(phosphatidylcholine), PE(phosphatidylethanolamine), PG(phosphatidylglycerol), PS(phosphatidylserine), SQDG(sulfoquinovosyldiacylglycerol).
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The cytochrome c oxidase enzyme from the Rhodobacter sphaeroides bacteria exists as a complex of four peptide subunits, two hemes, and a variety of lipids and metal ions held together by non-covalent forces. While the native enzyme functions as an associated unit, this complex usually dissociates during MALDI- TOF analysis. Through the use of matri...
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Changes in lipid levels/profiles can reflect health status and diseases. Urinary lipidomics, thus, has a great potential in clinical diagnostics/prognostics. Previously, only chloroform and methanol were used for extracting lipids from the urine. The present study aimed to optimize lipid extraction and examine differential lipid classes obtained by...
Citations
... A singlycharged protonated molecular ion of subunit I is 903 higher than the value calculated from the amino acid sequence (63,147 Da). This difference is almost certainly due to six histidines attached to subunit I and a possible modification at the N-terminus [37]. For subunit IV, an observed m/z value of 5335 is close to predicted mass (5403 Da) of the 'short' form of subunit IV, which is predominant in CcO isolated from wild-type Rb. ...
... For subunit IV, an observed m/z value of 5335 is close to predicted mass (5403 Da) of the 'short' form of subunit IV, which is predominant in CcO isolated from wild-type Rb. sphaeroides [37]. The sequencepredicted molecular masses of subunits II and III are 32,931 and 30,172 Da, respectively. ...
... The observed mass for subunit II was 861 Da lower than its calculated mass (32,931 Da) while the observed mass for subunit III was close (only 144 Da lower) to its calculated mass (30,172 Da). These differences are likely due to the post-translational processing removal of amino-acids [37]. ...
Delipidation of detergent-solubilized cytochrome c oxidase isolated from Rhodobacter sphaeroides (Rbs-CcO) has no apparent structural and/or functional effect on the protein, however affects its resistance against thermal or chemical denaturation. Phospholipase A2 (PLA2) hydrolysis of phospholipids that are co-purified with the enzyme removes all but two tightly bound phosphatidylethanolamines. Replacement of the removed phospholipids with nonionic detergent decreases both thermal stability of the enzyme and its resilience against the effect of chemical denaturants such as urea. In contrast to nondelipidated Rbs-CcO, the enzymatic activity of PLA2-treated Rbs-CcO is substantially diminished after exposure to high (>4M) urea concentration at room temperature without an alteration of its secondary structure. Absorbance spectroscopy and sedimentation velocity experiments revealed a strong correlation between intact tertiary structure of heme regions and quaternary structure, respectively, and the enzymatic activity of the protein. We concluded that phospholipid environment of Rbs-CcO has the protective role for stability of its tertiary and quaternary structures.
... Remarkably little is known about the function of CL in R. sphaeroides. The interaction of R. sphaeroides cytochrome c oxidase (CcO) and CL is essential for the full activity of the enzyme (51)(52)(53)(54). However, a previous study demonstrated that a CL deficiency in R. sphaeroides does not impair the structure and function of CcO or cause any significant growth defects (26). ...
Importance:
Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid, cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in regulating R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to alter or prevent biofilm formation.
... Previous studies have shown that the peroxidase activity of COX is independent of the K-channel and that at high pH values, peroxidase activity is greater than oxidase activity. 41,57,58 Upon comparison of rates of peroxidase and oxidase in both the WT and the mutant (Figure 4), the ratios of the WT and the mutant are approximately the same (2.3-and 2.1-fold increases, respectively, upon addition of hydrogen peroxide). ...
... (C) SDS− PAGE comparing Ni 2+ -purified WT (lane 1), FPLC pure WT (lane 2), Ni 2+ pure TM (lane 3), FPLC pure TM (lane 4), and Ni 2+ pure I−II oxidase (lane 5). The double band for SUII shown in panels B and C is a result of post-translational proteolytic processing, which cleaves 25 amino acids in the N-terminus and 15 amino acids in the C-terminus as has been shown by Distler et al.58 ...
The catalytic core of cytochrome c oxidase consists of three subunits that are conserved across species. The N-terminus of subunit III contains three histidine residues (3, 7 and 10) that are surface exposed, have physiologically relevant pKa values, and are in close proximity to the mouth of the D-channel in subunit I. A triple histidine mutation (to glutamine) was created in Rhodobacter sphaeroides. The mutant enzyme retains 60% of wild-type activity. Absorbance during steady-state turnover indicates that electrons accumulate at heme a in the mutant, accompanied by accumulation of the oxoferryl intermediate. When reconstituted into liposomes, the mutant enzyme pumps protons at half the efficiency of the wild-type. Finally, the mutant exhibits a lower cytochrome c peroxidation rate. Our results indicate that the mutation lowers activity indirectly by slowing proton uptake through the D-channel and that the three histidine residues stabilize the interactions between subunit I and subunit III.
... While occupying similar spatial location among the different oxidases, the genes encoding for subunit IV are located at genetic loci apart from that region coding for the canonical subunits (Noor and Soulimane , 2012 ). Alternative forms of R. sphaeroides subunit IV arising from the presence of two translation start codons have been reported (Distler et al. , 2004 ). In our laboratory, a single form of subunit IV was found to co-purify together with the recombinant caa 3 oxidase from T. thermophilus cells even when this subunit is not included in the Table 2 Detergent screening for the caa 3 oxidase crystallization. ...
Abstract Aerobic respiration, the energetically most favorable metabolic reaction, depends on the action of terminal oxidases that include cytochrome c oxidases. The latter forms a part of the heme-copper oxidase superfamily and consists of three different families (A-, B- and C-types). The crystal structures of all families have now been determined, allowing a detailed structural comparison from evolutionary and functional perspectives. The A2-type oxidase, exemplified by the Thermus thermophilus caa3-oxidase, contains the substrate cytochrome c covalently bound to the enzyme complex. In this article, we highlight the various features of caa3-enzyme and provide a discussion of their importance, including the variations in the proton and electron transfer pathways.
... Despite the drastically altered lipid profiles observed under these conditions, the bacteria are still viable and exhibit unhampered expression of a fully active four-subunit membrane protein complex of the terminal enzyme of the respiratory electron transport chain, cytochrome c oxidase (CcO) [11] . Notably, a number of lipid molecules have previously been demonstrated to be copurified with CcO, and highly conserved lipid binding sites have been identified crystallographically in the core subunits of CcO isolated from R. sphaeroides and bovine heart1314151617, suggesting an important functional role for lipids in CcO [18,19], as well as a requirement for lipids for achieving high-resolution CcO crystal structures. Importantly, abundant SQDG lipids have recently been found within the CcO enzymes purified from both the wild type R. sphaeroides as well as a cardiolipin-deficient mutant of R. sphaeroides (formed by disruption of the cardiolipin synthase (cls) gene), under normal and phosphate deficient growth conditions, and were identified for the first time in CcO crystals grown from the wild type and cardiolipin-deficient R. sphaeroides grown under normal phosphate conditions [11,12]. ...
Sulfoquinovosyldiacylglycerol (SQDG) lipids, found in plants and photosynthetic bacteria, can substitute for phospholipids under phosphate limiting conditions. Here, various low-energy ion activation strategies have been evaluated for the identification and characterization of deprotonated SQDG lipids from a crude membrane lipid extract of Rhodobacter sphaeroides, using collision- induced dissociation - tandem mass spectrometry (CID-MS/MS) in either a triple quadrupole mass spectrometer or in a hybrid quadrupole ion trap-multipole mass spectrometer coupled with high resolution / accurate mass analysis capabilities. In the triple quadrupole instrument, using energy resolved CID-MS/MS experiments, the SQDG head group specific product ion at m/z 225 (C(6)H(9)O(7)S(-)), rather than m/z 81 (SO(3)H(-)), was determined to provide the greatest sensitivity for SQDG lipid detection, and is therefore the preferred `fingerprint' ion for the identification of this lipid class from within complex lipid mixtures when using precursor ion scan mode MS/MS experiments. A comparison of conventional ion trap CID-MS/MS and -MS(n), with `low Q' CID-MS/MS, pulsed Q dissociation (PQD)-MS/MS and higher energy collision induced dissociation (HCD)-MS/MS performed in an LTQ Orbitrap Velos mass spectrometer, revealed that HCD-MS/MS coupled with high resolution/accurate mass analysis represents the most sensitive, and perhaps most importantly the most specific strategy, for ion trap based identification and characterization of SQDG lipids, due to the ability to readily distinguish the SQDG head group specific product ion at m/z 225.0069 from other products that may be present at the same nominal m/z value. Finally, the mechanisms responsible for formation of each of the major product ions observed by low-energy CID-MS/MS of deprotonated SQDG lipids were elucidated using uniform H/D exchange, HCD-MS/MS and high resolution mass analysis. Formation of the m/z 225 `fingerprint' ion occurs via a charge-remote cis-elimination reaction, likely involving transfer of a hydrogen from the hydroxyl group located on the C2 position of the sugar ring.
Completely revised and updated, this text provides an easy-to-read guide to the concept of mass spectrometry and demonstrates its potential and limitations. Written by internationally recognised experts and utilising "real life" examples of analyses and applications, the book presents real cases of qualitative and quantitative applications of mass spectrometry. Unlike other mass spectrometry texts, this comprehensive reference provides systematic descriptions of the various types of mass analysers and ionisation, along with corresponding strategies for interpretation of data. The book concludes with a comprehensive 3000 references. This multi-disciplined text covers the fundamentals as well as recent advance in this topic, providing need-to-know information for researchers in many disciplines including pharmaceutical, environmental and biomedical analysis who are utilizing mass spectrometry.
The gas-phase fragmentation reactions of various anionic adducts of phosphatidylcholine lipids have been examined by electrospray ionization and multistage tandem mass spectrometry (MSn) in a linear quadrupole ion trap mass spectrometer. The MS/MS and MSn fragmentation reactions of the anionic adducts were found to be highly dependent on the identity of the anions (A). Consistent with previous reports, MS/MS of phosphatidylcholine lipid ions formed by anionic adduction with formate, acetate and chloride resulted in a dominant product ion corresponding to the loss of (A + CH3) from the [M + A]− precursor ions. In contrast, MS/MS of other anion adducted precursor ions gave rise to multiple products, including [M + A(A + CH3)]−, [M + A(AH)]−, [M + A(AH + CH3)]−, [M + A(AH + (CH3)3N)]−, [M + A(A + (CH3)3NCHCH2)]− and [M + A(CH3)3N)]−. In order to gain evidence for the structures of these product ions, and the mechanisms by which they were formed, each of the products formed by MS/MS were subjected to further analysis by MS3 and MS4. The MS/MS and MSn fragmentation reactions of several deuterium isotope labeled phosphatidylcholine lipid standards, as well as authentic or structurally analogous lipid standards corresponding to the proposed product ion structures were also examined to gain evidence to support the proposed mechanisms. The results from this study indicate that great care must be taken to control the identity of the anions employed during lipid isolation, purification and separation, in order to allow the development and application of sensitive and robust neutral loss scan methods for mass spectrometry based lipid identification and quantitative analysis.
Many recent studies highlight the importance of lipids in membrane proteins, including in the formation of well-ordered crystals. To examine the effect of changes in one lipid, cardiolipin, on the lipid profile and the production, function, and crystallization of an intrinsic membrane protein, cytochrome c oxidase, we mutated the cardiolipin synthase (cls) gene of Rhodobacter sphaeroides, causing a >90% reduction in cardiolipin content in vivo and selective changes in the abundances of other lipids. Under these conditions, a fully native cytochrome c oxidase (CcO) was produced, as indicated by its activity, spectral properties, and crystal characteristics. Analysis by MALDI tandem mass spectrometry (MS/MS) revealed that the cardiolipin level in CcO crystals, as in the membranes, was greatly decreased. Lipid species present in the crystals were directly analyzed for the first time using MS/MS, documenting their identities and fatty acid chain composition. The fatty acid content of cardiolipin in R. sphaeroides CcO (predominantly 18:1) differs from that in mammalian CcO (18:2). In contrast to the cardiolipin dependence of mammalian CcO activity, major depletion of cardiolipin in R. sphaeroides did not impact any aspect of CcO structure or behavior, suggesting a greater tolerance of interchange of cardiolipin with other lipids in this bacterial system.
Many membrane proteins selectively bind defined lipid species. This specificity has an impact on correct insertion, folding, structural integrity and full functionality of the protein. How are these different tasks achieved? Recent advances in structural research of membrane proteins provide new information about specific protein-lipid interactions. Tightly bound lipids in membrane protein structures are described and general principles of the binding interactions are deduced. Lipid binding is stabilized by multiple non-covalent interactions from protein residues to lipid head groups and hydrophobic tails. Distinct lipid-binding motifs have been identified for lipids with defined head groups in membrane protein structures. The stabilizing interactions differ between the electropositive and electronegative membrane sides. The importance of lipid binding for vertical positioning and tight integration of proteins in the membrane, for assembly and stabilization of oligomeric and multisubunit complexes, for supercomplexes, as well as for functional roles are pointed out.
Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo(3) oxidase. The optimized protocol for expression and purification yields approximately 5 mg of the enzymatically active, uniformly (13)C,(15)N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The (13)C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo(3) oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly (13)C,(15)N-labeled membrane protein.
