Top PDF Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

molecules above them. The side chain of nearby Ser382 (Fig. 1) hydrogen-bonds to the −OH of the hydroxyethylfarnesyl chain of heme a. In the FR structure, however, this H bond is broken and Ser382 moves such that a cavity is formed that has been pro- posed to allow water to enter, and hence to allow proton transfer from the N phase via His413 into the middle region (23, 26). Indeed, in C1 simulations carried out with the oxidized form of CcO, the hydrogen bond between the −OH groups of Ser382 and the hydroxyethylfarnesyl chain of heme a persists, as do the two closest waters that are above Ser461/Ser458 (Fig. 4). For C2 simulations, this hydrogen bond tends to dissociate in both P-type catalytic intermediate states or with the BNC reduced (Fig. 4). Hence, although this dissociation is consistent with the structure of the FR state, the data show that dissociation of the H bond is not specifically correlated with any significant addi- tional hydration of the cavity between His413 and Ser461. In all simulations that failed to increase hydration in this region, the common feature instead was that all had His413 in its neutral state; in all of these, the low hydration persists and the occupancy of the two water sites above and below His413 does not fluctuate much (snapshots in Fig. 3 A and C), including in those cases where the BNC metals are reduced (C2-VII and C2-VIII). To further test this behavior, two ∼1-μs MD simulations were per- formed in the FR state with the C3 system (Table 3). Again, hydration remained minimal in the regions immediately above and below His413 in these long-time-scale simulations, with only 1.6 ± 1.1 and 1.3 ± 0.8 water molecules within 5 Å of His413 and Ser461 in two simulation replicas. These conclusions are also supported by data in Fig. 5 and SI Appendix, Tables S3 and S6 , which show that this low water occupancy persists throughout the simulation times in all redox states studied for both C1 and C2 systems, provided that the His413 is neutral. This suggests that there is a permanent barrier to proton transfer in this region, regardless of the Ser382 conformation, in contrast to proposals Model system Cu A Heme a BNC* His413 Asp51 Glu242 Tyr244 Lys319 Asp364
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Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

molecules above them. The side chain of nearby Ser382 (Fig. 1) hydrogen-bonds to the −OH of the hydroxyethylfarnesyl chain of heme a. In the FR structure, however, this H bond is broken and Ser382 moves such that a cavity is formed that has been pro- posed to allow water to enter, and hence to allow proton transfer from the N phase via His413 into the middle region (23, 26). Indeed, in C1 simulations carried out with the oxidized form of CcO, the hydrogen bond between the −OH groups of Ser382 and the hydroxyethylfarnesyl chain of heme a persists, as do the two closest waters that are above Ser461/Ser458 (Fig. 4). For C2 simulations, this hydrogen bond tends to dissociate in both P-type catalytic intermediate states or with the BNC reduced (Fig. 4). Hence, although this dissociation is consistent with the structure of the FR state, the data show that dissociation of the H bond is not specifically correlated with any significant addi- tional hydration of the cavity between His413 and Ser461. In all simulations that failed to increase hydration in this region, the common feature instead was that all had His413 in its neutral state; in all of these, the low hydration persists and the occupancy of the two water sites above and below His413 does not fluctuate much (snapshots in Fig. 3 A and C), including in those cases where the BNC metals are reduced (C2-VII and C2-VIII). To further test this behavior, two ∼1-μs MD simulations were per- formed in the FR state with the C3 system (Table 3). Again, hydration remained minimal in the regions immediately above and below His413 in these long-time-scale simulations, with only 1.6 ± 1.1 and 1.3 ± 0.8 water molecules within 5 Å of His413 and Ser461 in two simulation replicas. These conclusions are also supported by data in Fig. 5 and SI Appendix, Tables S3 and S6 , which show that this low water occupancy persists throughout the simulation times in all redox states studied for both C1 and C2 systems, provided that the His413 is neutral. This suggests that there is a permanent barrier to proton transfer in this region, regardless of the Ser382 conformation, in contrast to proposals Model system Cu A Heme a BNC* His413 Asp51 Glu242 Tyr244 Lys319 Asp364
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The H channel is not a proton transfer path in yeast cytochrome c oxidase

The H channel is not a proton transfer path in yeast cytochrome c oxidase

A B S T R A C T Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site- directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electro- static interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.
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The H channel is not a proton transfer path in yeast cytochrome c oxidase

The H channel is not a proton transfer path in yeast cytochrome c oxidase

A B S T R A C T Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site- directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electro- static interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.
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Redox-coupled proton pumping in cytochrome c oxidase: Further insights from computer simulation

Redox-coupled proton pumping in cytochrome c oxidase: Further insights from computer simulation

Received 15 August 2007; received in revised form 12 November 2007; accepted 20 November 2007 Available online 4 December 2007 Abstract The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a 3 (PRDa 3 ) for the first time. The results from these molecular dynamics simulations indicate that the PRDa 3 can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa 3 side chain isomerization and the proton translocation between Glu242 and the PRDa 3 site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa 3 site.
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Enhanced Cytochrome C Oxidase Activity in WBC of COPD Patients

Enhanced Cytochrome C Oxidase Activity in WBC of COPD Patients

pulmonary resistance and hyperinflation. Cytochrome C Oxidase (COX) as a key oxidative enzyme modulates oxygen uptake and catalyzes the oxidation of reduced cytochrome C by molecular oxygen. In vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen. Thus, a better understanding of the role of COX in patients with COPD can provide an important link between the availability of oxygen to tissues and the regulation of oxygen uptake and energy production in these patients.

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Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production

Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production

MT1131 and the previously sequenced SB1003 strain (19) indeed defined the crtD121 mutation (C-to-T change at position 753,947 in crtD121 [RCC00683]) in R. capsulatus MT1131 and also re- vealed the presence of ~1,260-bp deletion (from positions 3,211,370 to 3,212,630) next to a transposase gene, as well as ~385 SNPs and indels across the whole genome (see Table S3 in the supplemental material and http://networks.systemsbiology.net /rcaps/). As expected, the genetically constructed deletion- insertion (starting at position 2,366,510) to knock out ccoA was present in SE8 and SE8Ri genomes. Pairwise comparison of the SNPs between all strains showed only one difference for SE8R2 (in the intergenic region upstream of RCC02563-hypothetical pro- tein), SE8R5 (in RCC01565) and SE8R6 (in RCC01100) (Ta- ble S3). However, when the indels were compared, a single differ- ence in the RCC01180 gene (at position 1,253,275) annotated as “copA, copper translocating P-type ATPase” was found between the wild type (MT1131) and the ⌬ccoA mutants (SE8E/SE8H) versus the ccoA revertants (SE8R1, -2, -5, and 6) (Fig. 3A). This indel corresponded to an addition of a single cytosine residue in SE8R1A/B and SE8R2 (copA11C ⌬ ccoARev group 1), and a dele- tion of a single cytosine residue in SE8R5 and SE8R6 (copA9C ⌬ ccoARev group 2), in the same stretch of 10 consecutive cytosine residues between base pairs 231 to 241 of RCC01180 (called copA hereafter) (Fig. 3B). PCR amplification of this region from the genomic DNA from appropriate strains combined with sequenc-
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Structure/function of oxygen regulated isoforms in cytochrome c oxidase

Structure/function of oxygen regulated isoforms in cytochrome c oxidase

Previous studies have revealed that yeast cells require a subunit V isoform for a functional holocytochrome c oxidase and that Va and Vb are interchangeable (Trueblood and Poyton, 1987); that is, either isoform can function in the holoenzyme. These findings suggest that, in addition to the differential functions mentioned above, these two isoforms have conserved functions as well. From a comparison of their primary sequences, it is clear that there are regions of perfect homology in these two polypeptides. One of these is at their carboxyl termini. Recently, we have analyzed several point mutations in COX5a (P. V. Burke and R. O. Poyton, in preparation). One of these, cox5a-1, is a nonsense mutation at residue 124 in the carboxyl-terminal domain; this deletes the ten carboxyl-terminal amino acids of the protein and leads to complete loss of activity. Another is a revertant that replaces the stop codon at residue 124 with a serine and, in so doing, restores partial activity. This revertant, with Ser124 replacing Trp124, supports approximately half the level of activity of the wild-type protein. Together, these two mutants establish that the ten carboxyl-terminal amino acids, which are completely conserved between Va and Vb, are essential.
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Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core

Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core

Over the last 20 years, mutant screen strategies have been also used in humans, since defective COX biogenesis results in devastating human mitochondrial diseases frequently involving brain, skeletal muscle and heart tissues (reviewed in [ 11, 21, 43 –45 ]). To date, with the double exception of an infantile encephalomyopathy caused by a mu- tation in the nuclear encoded subunit COX6B1 [46] and an exocrine pan- creatic insuf ficiency caused by a mutation in the COX4I2 gene [47] , all Mendelian disorders presenting COX de ficiency have been assigned to mutations in ancillary factors. Speci fically, mutations have been found in SURF1, required for the formation of early assembly intermediates [48, 49] , SCO1 and SCO2, required for COX copper metallation [50 −54] , COX10 and COX15, essential for heme A biosynthesis [55-57] , and finally in LRPPRC [58] and TACO1 [59] , required for the expression of COX sub- units. Mutant fibroblast cell lines from patients suffering from these disor- ders have been used to study the accumulation of subassembly intermediates in the absence of speci fic COX assembly factors and obtain information concerning the assembly step either catalyzed or affected by the mutated factor [31, 60, 61] . These studies have also provided informa- tion concerning mammalian speci fic COX biogenetic factors, such as TACO1, an evolutionary conserved protein although it functions as a mito- chondrial COX1 translational activator speci fically in mammals [59] .
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Pet191 Is a Cytochrome c Oxidase Assembly Factor in Saccharomyces cerevisiae

Pet191 Is a Cytochrome c Oxidase Assembly Factor in Saccharomyces cerevisiae

residues are shown in black. (C) Mitochondria isolated from pet191 ⌬ cells carrying an empty vector, PET191, or its mutant forms were subjected to SDS-PAGE followed by Western blot analysis with the antibodies against the Myc epitope and porin. (D) WT cells overexpressing PET191 or its C5A, C15A, or C56A mutant versions were serially diluted, plated as described for panel A, and grown for 3 days at 30°C. (E) Mitochondria from the cells coexpressing Pet191-HA and Pet191-Myc or its C5A mutant form were solubilized and analyzed as described above, except that DTT treatment was not included. Collected fractions were analyzed using anti-Myc and anti-HA antibodies. (F) Mitochondria isolated from the WT PET191::3HA strain with (lanes 1 to 3) or without (lanes 4 to 6) Pet191-Myc were lysed, and clarified extracts were immunoprecipitated with agarose-coupled anti-Myc antibodies. Co-IP of Pet191-Myc and Pet191-HA was performed as described previously (20), using anti-Myc–agarose- coupled beads (Santa Cruz Biotechnology). The load (representing 5% of the extracts; lanes 1 and 4), entire-wash (lanes 2 and 5), and eluate (lanes 3 and 6) fractions were analyzed by immunoblotting.
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Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver

Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver

The higher Kp values and the decrease of Kp due to KCN addition in the presence of LM indicate that LM contain higher levels of intrinsic peroxidases than BM, confirming previous studies [27]. It has been reported that BM contains bilirubin oxidase, which also has been detected in other organs including liver [28,29]. Other peroxidases known to be present in mitochondria include phospholipid hydroperoxide glutathione peroxidase (PHGPx) [30], glutathione peroxidase (GPx), catalase (CAT) [30,31], and peroxiredoxin (Prx) III [32], which like other hemoproteins, are inhibitable by KCN [29,31]. In LM, these intrinsic mitochondrial peroxidases contrib- ute to the UCB oxidation measured in the presence of the added HRP, accounting for the almost 2× higher apparent Kp in the presence of LM compared to BM. These find- ings emphasize the importance of determining Kp in complete systems containing the organelles being stud- ied.
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On the Conformational Dynamics of DNA: A Perspective from Molecular Dynamics Simulations

On the Conformational Dynamics of DNA: A Perspective from Molecular Dynamics Simulations

RXRα (A) and the RXRβ (B). Arrows show regions of high occupancy surrounding the ligand. Only part of the binding pocket is shown with the side chain of Ile drawn as licorice for reference. For clarity, the overlays of the averaged binding position of bexarotene in the separate MD studies are shown as sticks; bexarotene was not present in the molecular flooding simulations, however.

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Effects of oxidation on tensile deformation of iron nanowires: Insights from reactive molecular dynamics simulations

Effects of oxidation on tensile deformation of iron nanowires: Insights from reactive molecular dynamics simulations

onto the Fe core and did not exhibit any cracks during the tensile loading. The onset of plasticity for the [001]-oriented pure and oxide-coated Fe NWs, promoted by specifically nucleation through a/6h111i Shockley partial dislocation on the {112} glide planes from the energetically favorable edge on the free surface of the cylindrical NW, 31 is clearly seen from the stress-strain curve shown in Figs. 7 and 8 and is associated with a sharp decline in stress from the maximum critical yield stress to 0.0 GPa. However, for the oxide- coated Fe NWs, yielding occurs at relatively lower strain and stress values compared to those of the pure NW, i.e., the defects due to the oxide shell layer thickness increase and the mechanical resistance to plastic deformation decreases. When the applied strain is increased further, the a/6h111i Shockley partial dislocation on the {112} glide plane starts to propagate laterally toward the other NW surface along the cross-sectional length, and the new partial dislocation quickly emerges from the next parallel {112}-glide plane. 31 Surprisingly, the crystallographic lattice reorientation imme- diately begins with the formation of these two partial dislo- cations. Consequently, a complete and unique abrupt structural reorientation from [001] to [110] driven defect is observed between these two partial dislocations. Moreover, nucleation of two close and identical partial dislocations is activated on the {112}-glide plane in the h111i direction without the influence of native oxide layer effects, as shown in Figs. 7 and 8 ( supplementary material , Fig. 1 ). As a result of consecutive continuous motion of partial dislocations from one surface toward the opposite side on the adjacent {112} glide planes, the partial dislocations finally coincide at the opposite surface of the NWs, where they are annihilated, leaving behind a pair of stacking faults with continued ten- sile loading. 31 The common neighbor analysis (CNA) tech- nique with the visualization package tool OVITO was employed to accurately identify and characterize the NW defect structure. 59 Defects associated with the local crystal- line disordered atoms, distributed at the surface, dislocation core, and twin boundaries, are clearly shown in Figs. 7 and
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Polymer Nanocomposites: Insights from Theory and Molecular Simulations.

Polymer Nanocomposites: Insights from Theory and Molecular Simulations.

Little is known about the impact of solvent or additives on the morphological parameters and interfacial energetics. More studies are required to know whether we can control the morphology and interfacial energetics with use of different solvents and how this influence can affect adsorption of polymer on inorganic metal surfaces and conformation of the ligands and polymer chains as a function of solubility of the components, orientation of nanoparticles. In the present study we have discussed PNCs based upon three diverse application areas like redox active core dendrimer, nanocomposite polymer solar cell and metal nanoparticle polymer composite material. We study the role of solvent, impact of solubility on the morphology of polymer nanocomposite, role of orientation of anisotropic nanoparticles in organic solar cells devices and how solvent can change the dendritic architecture of redox core dendrimers with use of quantitative and thermodynamics measures with atomistic molecular dynamics simulations. We anticipate that our results will help in optimization of experimental parameters in current applications and the emergence of new techniques can be facilitated by a better theoretical understanding of the relation between resultant polymer thin film properties, such as composition and morphology, and the materials and fabrication parameters.
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Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus

Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus

[118] , is a heme handling protein (HHP) like CcmC [106] . CcmI is a bipartite protein that contains a membrane embedded N-terminal region with two transmembrane helices and a cytoplasmic leucine- zipper-like motif containing loop (CcmI-1 domain) and a large peri- plasmic C-terminal extension with tetratricopeptide repeat (TPR)- like motifs (CcmI-2 domain) [119 –121] . Unlike most other bacteria, in E. coli the homologue of CcmI-2 domain is fused to the C-terminal end of CcmH, leaving only CcmF and a modi fied “CcmH” as compo- nents of heme ligation core complex [122] . R. capsulatus, mutants lacking CcmI can be suppressed by overproduction of CcmF and CcmH or CcmG. Complementation studies with two distinct domains of CcmI indicated that CcmF and CcmH overproduction relates to the
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Coordination of cytochrome c oxidase gene expression in the remodelling of skeletal muscle

Coordination of cytochrome c oxidase gene expression in the remodelling of skeletal muscle

In any multistep process, some steps will be more important in determining the overall rate. In the case of metabolic pathways, quantitative control analyses can assign a step a control coefficient, or, in more qualitative terms, some enzymes are considered to be ‘rate limiting’ or ‘rate determining’. These steps are often the slowest, and are typically subject to other forms of metabolic regulation. It is reasonable to speculate that some COX subunits may be more limiting than others and, if so, may have a greater role in determining changes in COX activity. By analogy with metabolic pathways, rate-determining subunits might be those where changes in mRNA level best paralleled COX activity. Many studies have used molecular genetic interventions to impose a reduction in the synthesis of individual subunits to assess the effects on COX biosynthesis. For example, dramatic reductions in COX arise when COX5A is disrupted using morpholinos in zebrafish (Baden et al., 2007) and RNAi in Caenorhabditis elegans (Suthammarak et al., 2009). Likewise, loss of COX activity is seen when COX6A synthesis is disrupted in a mouse knockout (Radford et al., 2002), Drosophila mutants (Liu et al., 2007) and human cell line knockouts (Fornuskova et al., 2010). Reductions in COX4 lead to a decrease in COX content in mammalian lines (Li et al., 2006; Fornuskova et al., 2010) and C. elegans (Suthammarak et al., 2009). In some cases, this intervention leads to the accumulation of assembly intermediates and enzymatic abnormalities (Fornuskova et al., 2010). Such studies are used to assess the importance of subunits in assembly or function but they are not intended to explore constraints on the rate of synthesis during adaptive remodelling. The question of whether any of these genes exert disproportionate control over COX synthesis has not been addressed experimentally. This study gives an indication of the sensitivity of COX synthesis to variation in subunit expression. Thus, we consider these data to
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A study of cytochrome C oxidase subunit 1 polymorphism in Malay population

A study of cytochrome C oxidase subunit 1 polymorphism in Malay population

Mitochondria are organelles which is producing energy inside the cells. The energy will be produced through oxidative phosphorylation process by converting the fuels into ATP (Taanman; 1998). Mitochondria have a separate genome from nucleus genome which is called mitochondrial DNA (mtDNA). mtDNA is placed in the mitochondrial matrix. There are several copies of mtDNA in each mitochondrion organelle in mammalian cells (Michaels et al; 1982). MtDNA is small and has high copy number inside the cell and it will be easier to isolate the DNA from this organelle therefore, the first genome sequencing projects have been done on this molecule (Http://megasun.bch.umontreal.ca). The sequencing projects of mtDNA molecule have been done in the huge number of species and also in human (Anderson et al; 1981).
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Proton transfer pathways in the mitochondrial S  cerevisiae cytochrome c oxidase

Proton transfer pathways in the mitochondrial S cerevisiae cytochrome c oxidase

bilization the membranes were diluted to 2 mg/ml in buffer (50 mM KPi, 100 mM KCl and 1.5% n-dodecyl β -D-maltoside (DDM (w/v)) and solubilized for 1 hour. To remove cyt. c the supernatant from the centrifugation step after solubilization was run over a column loaded with a cation-exchanger (Bio-Rex 70, Bio-Rad) equili- brated with 50 mM KPi, 100 mM KCl and 0.035% DDM. After addition of 5 mM imidazole to the flow-through from the ion-exchanger it was loaded on a column filled with 25 ml Ni-resin (NI Sepharose 6 Fast Flow from GE healthcare), pre-equilibrated with (20 mM KPi, 150 mM KCl and 0.035% DDM). The column was washed with four column volumes of wash buffer (20 mM KPi, 150 mM KCl, 10 mM imidazole and 0.035% DDM). This was followed in time by a two-step elution of four column volumes at each imidazole concentration (20 mM KPi, 150 mM KCl, 40 or 100 mM imidazole and 0.035% DDM). The eluted fractions were pooled and concentrated in a 100 kDa cut-off filter (Merck Millipore). Buffer exchange (elution buffer without imidazole) was performed to lower the imidazole content to sub-µM concentrations.
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Leigh's Encephalomyelopathy in a Patient With Cytochrome c Oxidase Deficiency in Muscle Tissue

Leigh's Encephalomyelopathy in a Patient With Cytochrome c Oxidase Deficiency in Muscle Tissue

carboxylase activity in liver tissue was shown by several investigators in children with SNE proven by autopsy.3’4 Yoshida et al.5 suggested that abnor- mally high levels of pyruvate and[r]

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Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

In general, the proton-pumping stoichiometry is presumably determined by the relative proton-transfer rates from Glu286 to the catalytic site and the pump site, respectively. These rates are presumably dependent on the local environment of the Glu286, not only because changes in the environment would alter the proton connectivity, but more importantly because the Glu286 presumably has to isomerize during the transfer. Such an isomerization would be sensitive to any changes in the hydrogen-bonding pattern of the Glu. A structural isomerization of the Glu286 side chain, linked to proton transfer was originally proposed by Iwata et al. [10] based on an inspection of the P. denitri ficans CytcO crystal structure (because of the lack of protonic connectivity between the Glu and the catalytic site or the pump site). This proposal is supported by results from theoretical calculations [50,151 –155] , which suggested that Glu286 conforma- tional isomerization may be involved in proton gating. Results from more recent computational studies suggested that Glu286 may function as a proton valve, which can adopt distinct conformations thereby controlling the proton connectivity to solution, the catalytic site and the pump site [156,157] . However, as pointed out by Warshel and colleagues [54] , an isomerization of a side chain by itself cannot establish a gate (because of microscopic reversibility). Nevertheless, changes in the equilibrium constant between the Glu286 conformers are expected to result in proton leaks that would decrease the proton- pumping stoichiometry [157] .
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