Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.9.3.1 (cytochrome oxidase)
8,822 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peroxynitrite anion, the reaction product of superoxide and nitric oxide, is a potent biological oxidant, which inactivates mammalian heart mitochondrial NADH-coenzyme Q reductase (complex I), succinate dehydrogenase (complex II), and ATPase, without affecting cytochrome c oxidase (complex IV). In this paper, we evaluated the effect of peroxynitrite on mitochondrial membrane integrity and permeability under low calcium concentration. Phosphate buffer was used in most of our experiments since Hepes, Tris, mannitol, and sucrose were found to inhibit the oxidative chemistry of peroxynitrite. Peroxynitrite (0.1-1.0 mM) caused a dose-dependent decrease in the ability of mitochondria to build up a membrane potential when N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate were used as substrate. Elimination of the membrane potential was accompanied by penetration of the osmotic support (KCl/NaCl) into the matrix as judged by the parallel occurrence of mitochondrial swelling. This swelling was partially inhibited by dithiothreitol (DTT) or butylated hydroxytoluene (BHT) and was insensitive to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ADP, and cyclosporin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicated that alterations in membrane permeability were associated with the production of protein aggregates due to membrane protein thiol cross-linking. The protective effect of DTT on both mitochondrial swelling and protein polymerization suggests the involvement of disulfide bonds in the membrane permeabilization process. In addition, the increase in thiobarbituric acid-reactive substances and the partial inhibitory effect of BHT indicate the occurrence of lipid peroxidation. These results support the idea that under our experimental conditions peroxynitrite causes mitochondrial structural and functional alterations by Ca2+-independent mechanisms through lipid peroxidation and protein sulfhydryl oxidation.
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PMID:Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation. 930 96

Cardiolipin synthase catalyzes the synthesis of the mitochondrial phospholipid cardiolipin. Cardiolipin synthase is a unique membrane-bound enzyme in that it utilizes two phospholipids, both insoluble in water, as substrates. Kinetic analysis suggests that the enzyme forms a ternary complex with the two lipid substrates, and that a divalent metal ion directly associates with cardiolipin synthase to form the active enzyme. While little is known about the regulation of cardiolipin synthase in yeast, activity is reduced in mutants in which the mitochondrial genome is deleted, and in mutants with defective respiratory complexes. In p0 mutants, which contain no mitochondrial DNA and are defective in the assembly of many mitochondrial membrane protein complexes, cardiolipin synthase activity is reduced by 50%. Mutants defective in respiratory complexes, particularly those incapable of cytochrome oxidase assembly, also have reduced cardiolipin synthase activity. Thus it is likely that respiration and cardiolipin formation are interdependent. The enzyme was recently purified from the budding yeast Saccharomyces cerevisiae. Enzyme activity was associated with a 25-30-kDa protein. The amino acid sequence of this protein, combined with the availability of the complete yeast genome sequence, will hopefully lead to the identification of the structural gene for this enzyme in the near future.
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PMID:Cardiolipin synthase from yeast. 937 Mar 34

The cytochrome aa3 (600 nm) complex, or menaquinol oxidase, from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory membrane protein complexes. We have characterized some spectral properties of this enzyme and its reaction with cyanide. The magnetic circular dichroism (MCD) spectrum of the oxidized enzyme has a single band at 1560 nm in the near-infrared region assigned to bis-histidine-ligated, low-spin ferricytochrome a. The other heme, cytochrome a3, is presumably high-spin in the oxidized enzyme, as isolated. The absence of a trough in the MCD spectrum at 790 nm, observed previously with mammalian cytochrome c oxidase and assigned to CuA (Greenwood et al., Biochem. J. 215, 303-316, 1983), is consistent with the absence of this center from the menaquinol oxidase. When the heme ligand cyanide is added to oxidized menaquinol oxidase, a new MCD band appears at 2010 nm, while the band at 1560 nm is unperturbed. The new band is assigned to low-spin ferricytochrome a3 bound with cyanide. The long-wavelength position of this cyanide-induced band is proposed to arise from the close interaction of cytochrome a3 with the copper atom, CuB. The kinetics of cyanide binding to oxidized cytochrome aa3(600 nm) reveal a spectrally simple, yet kinetically complex process. The reaction is biphasic with second-order rate constants of 45 and 0.61 M-1s-1 at 1 mM KCN, with each phase constituting about 50% of the overall reaction. When the enzyme is subjected to a cycle of anaerobic reduction and air oxidation, the subsequent reaction with cyanide occurs in a single phase at the faster rate. This behavior is ascribed to different conformations of the binuclear center exhibiting different reactivities with cyanide, and is in keeping with that previously established for the structurally more complex mitochondrial cytochrome c oxidase. However, the electronic spectral characteristics of some of the species involved in these reactions are different in the present bacterial case from those of reported eukaryotic systems.
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PMID:Spectral and cyanide binding properties of the cytochrome aa3 (600 nm) complex from Bacillus subtilis. 947 2

The preparation of pure and homogeneous membrane proteins or membrane protein complexes is time consuming, and the yields are frequently insufficient for structural studies. To circumvent these problems we established an indirect immunoaffinity chromatography method based on engineered Fv fragments. cDNAs encoding the variable domains of hybridoma-derived antibodies raised against various membrane proteins were cloned and expressed in Escherichia coli. The Fv fragments were engineered to serve as bifunctional adaptor molecules. The Fv fragment binds to the epitope of the membrane protein, while the Strep tag affinity peptide, which was fused to the carboxy-terminus of the VH chain, immobilizes the antigen-Fv complex on a streptavidin sepharose column. The usefulness of this technique is illustrated with membrane protein complexes from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), and subcomplexes or individual subunits thereof. These membrane proteins were purified simply by combining the crude P. denitrificans membrane preparation with the E. coli periplasmic cell fraction containing the corresponding Fv fragment, followed by solubilization and streptavidin affinity chromatography. Pure and highly active membrane protein complexes were eluted in the Fv-bound form using diaminobiotin for mild competitive displacement of the Strep tag. The affinity column could thus be reused under continuous operation for several months. Five to 10 mg of membrane protein complexes could be obtained without any detectable impurities within five hours.
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PMID:Engineered Fv fragments as a tool for the one-step purification of integral multisubunit membrane protein complexes. 963 56

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.
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PMID:Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency. 971 4

Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants. We have isolated several independent suppressors that compensate for the absence of Oxa1p. Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor. Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity. The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity. Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity. In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains. We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p.
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PMID:Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1. 975 93

The machinery that inserts mitochondrially encoded proteins into the inner membrane and translocates their hydrophilic domains through the membrane is poorly understood. We have developed a genetic screen for Saccharomyces cerevisiae mutants defective in this export process. The screen is based on the fact that the hydrophilic polypeptide Arg8(m)p is exported from the matrix if it is synthesized within mitochondria as a bifunctional Cox2p-Arg8(m)p fusion protein. Since export of Arg8(m)p causes an Arg(-) phenotype, defective mutants can be selected as Arg(+). Here we show that mutations in the nuclear gene PNT1 block the translocation of mitochondrially encoded fusion proteins across the inner membrane. Pnt1p is a mitochondrial integral inner membrane protein that appears to have two hydrophilic domains in the matrix, flanking a central hydrophobic hairpin-like anchor. While an S. cerevisiae pnt1 deletion mutant was more sensitive to H(2)O(2) than the wild type was, it was respiration competent and able to export wild-type Cox2p. However, deletion of the PNT1 orthologue from Kluyveromyces lactis, KlPNT1, caused a clear nonrespiratory phenotype, absence of cytochrome oxidase activity, and a defect in the assembly of KlCox2p that appears to be due to a block of C-tail export. Since PNT1 was previously described as a gene affecting resistance to the antibiotic pentamidine, our data support a mitochondrial target for this drug.
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PMID:Mutations affecting a yeast mitochondrial inner membrane protein, pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix. 1049 May 99

The catalytic core of cytochrome c oxidase is composed of three subunits: I, II, and III. Subunit III is a highly hydrophobic membrane protein that contains no redox centers; its role in cytochrome oxidase function is not obvious. Here, subunit III has been removed from the three-subunit mitochondrial-like oxidase of Rhodobacter sphaeroides by detergent washing. The resulting two-subunit oxidase, subunit III (-), is highly active. Ligand-binding analyses and resonance Raman spectroscopy show that its heme a(3)-Cu(B) active site is normal. However, subunit III (-) spontaneously and irreversibly inactivates during O(2) reduction. At pH 7.5, its catalytic lifetime is only 2% that of the normal oxidase. This suicide inactivation event primarily alters the active site. Its ability to form specific O(2) reduction intermediates is lost, and CO binding experiments suggest that the access of O(2) to reduced heme a(3) is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a(3); electron transfer between the hemes is inhibited. Ligand-binding experiments and resonance Raman analysis show that increased flexibility in the structure of the active site accompanies inactivation. Cu(B) is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of Cu(B) and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the Cu(B) center via long-range interactions.
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PMID:Suicide inactivation of cytochrome c oxidase: catalytic turnover in the absence of subunit III alters the active site. 1058 46

A search of the Bacillus subtilis genome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase. B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, Cu(A) and Cu(B), and a menaquinol oxidase that has only Cu(B). Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the DeltaypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the Cu(A) site of cytochrome c oxidase.
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PMID:Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis. 1083 75

The amino- and carboxy-terminal domains of mitochondrially encoded cytochrome c oxidase subunit II (Cox2p) are translocated out of the matrix to the intermembrane space. We have carried out a genetic screen to identify components required to export the biosynthetic enzyme Arg8p, tethered to the Cox2p C terminus by a translational gene fusion inserted into mtDNA. We obtained multiple alleles of COX18, PNT1, and MSS2, as well as mutations in CBP1 and PET309. Focusing on Cox18p, we found that its activity is required to export the C-tail of Cox2p bearing a short C-terminal epitope tag. This is not a consequence of reduced membrane potential due to loss of cytochrome oxidase activity because Cox2p C-tail export was not blocked in mitochondria lacking Cox4p. Cox18p is not required to export the Cox2p N-tail, indicating that these two domains of Cox2p are translocated by genetically distinct mechanisms. Cox18p is a mitochondrial integral inner membrane protein. The inner membrane proteins Mss2p and Pnt1p both coimmunoprecipitate with Cox18p, suggesting that they work together in translocation of Cox2p domains, an inference supported by functional interactions among the three genes.
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PMID:Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane. 1195 Sep 26


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