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Proton pumping by cytochrome oxidase as studied by time-resolved stopped-flow spectrophotometry.

The H+/e- stoichiometry for the proton pump of cytochrome c oxidase reportedly varies between 0 and 1, depending on experimental conditions. In this paper, we report the results obtained by a combination of transient optical spectroscopy with a time resolution of 10 ms and a singular value decomposition analysis to follow the kinetics, separate the observed spectral components, and quantitate the stoichiometry of the pump. By using cytochrome oxidase reconstituted into small unilamellar vesicles, we show that the time courses of ferrocytochrome c oxidation and phenol red acidification or alkalinization fit a simple kinetic scheme. The fitting procedure leads to unbiased and objective determination of the H+/e- ratio under various experimental conditions. The proton-pumping stoichiometry was found to be 1.01 +/- 0.10, independent of the number of turnovers, proton back-leak rate, or type of experiment (oxidant or reductant pulse).

Animals↗

Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates.

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3' to 5' editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3' to 5' patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3' to 5' editing of the COIII mRNA with incorrect guide RNA molecules.

Animals↗

Relationship among coelacanths, lungfishes, and tetrapods: a phylogenetic analysis based on mitochondrial cytochrome oxidase I gene sequences.

To clarify the relationship among coelacanths, lungfishes, and tetrapods, the amino acid sequences deduced from the nucleotide sequences of mitochondrial cytochrome oxidase subunit I (COI) genes were compared. The phylogenetic tree of these animals, including the coelacanth Latimeria chalumnae and the lungfish Lepidosiren paradoxa, was inferred by several methods. These analyses consistently indicate a coelacanth/lungfish clade, to which little attention has been paid by previous authors with the exception of some morphologists. Overall evidence of other mitochondrial genes reported previously and the results of this study equally support the coelacanth/lungfish and lungfish/tetrapod clades, ruling out the coelacanth/tetrapod clade.

Amino Acid Sequence↗

Organization of cytochrome oxidase staining in the visual cortex of nocturnal primates (Galago crassicaudatus and Galago senegalensis): I. Adult patterns.

The distribution and differential staining patterns of cytochrome oxidase (CO) activity in visual cortical areas have provided useful anatomical markers for the modular organization of area 17 (striate cortex) and area 18 in primates. In macaque and squirrel monkeys, previous studies have shown that the majority of cells that lie in areas of high CO activity are color selective, are nonoriented, and project to adjacent zones of high CO activity in area 17 and to stripes of high CO activity in area 18. By contrast, most cells in zones with weak CO activity in area 17 have relatively narrow orientation tuning and are not color selective (Livingstone and Hubel: J. Neurosci. 4:309-356, 2830-2835, '84; 7:3371-3377, '87). The periodic organization of CO activity in area 17, the "blobs," and the stripe-like organization in area 18 thus seem to define visual cortical processing modules and/or channels in primates. We have investigated the organization of CO activity in areas 17 and 18 in two species of nocturnal prosimian primates [Galago crassicaudatus (GCC) and Galago senegalensis (GSS)] in order to evaluate CO staining patterns in primates that have been reported to possess almost exclusively rod retinae and no color vision. In area 17 of both species, our results show that, as in diurnal and nocturnal simian primates, the darkest CO staining occurs in layers III and IV, with clear periodicity in layer III (i.e., CO blobs) and homogeneous staining in layer IV beta, the cortical recipient sublayer of the geniculate parvocellular layers. In GCC, individual blobs in layer III appear to be larger and less frequent than has been reported for the macaque monkey. Unlike simian primates, both galago species exhibit clear CO periodicities within layer IV alpha, the cortical recipient sublayer of the magnocellular geniculate layers. In addition, faint CO periodicities are apparent in layer VI and scattered large darkly CO stained pyramidal cells are visible throughout layer V. Quantitative analysis suggests that CO periodicities are more frequent in GSS than in GCC, suggesting that there may be evolutionary pressure to maintain the same number of CO modules within the smaller striate cortex of the lesser galago, although this is not the trend found across distantly related species. CO activity in area 18 is less well-developed than reported in other primates. In fact, we could not reliably identify discontinuities in CO staining in area 18 of GSS.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Resolving phylogeny at the family level by mitochondrial cytochrome oxidase sequences: phylogeny of carrion beetles (Coleoptera, Silphidae).

We investigated the phylogenetic relationships of carrion beetles (Coleoptera, Silphidae) using 2094 bp of their mitochondrial cytochrome oxidase subunit I and II and tRNA leucine gene sequences. Shorter fragments of this gene region previously have been used to establish generic relationships in insects. In this study, they provided more than sufficient resolution, although the third positions of the protein-coding sequences reached saturation for the deeper divergences. This first published phylogeny for the Silphidae comprises 23 species from 13 genera sampled across the geographic range of the family. In addition, we included species from three related families as outgroups. One of these families, the Agyrtidae, was, until recently, included in the Silphidae, but its resolution here justifies its current position as a separate family. The silphid subfamilies Nicrophorinae and Silphinae are monophyletic in all analyses. All genera for which several species were sampled are supported as monophyletic groups, with the exception of the genus Silpha. European and North American representatives of two Nicrophorus species described from both continents are supported as each others' closest relatives. The lineage that colonized Gondwanaland and that most likely originated in the Palearctic is the most basal within the Silphinae.

Animals↗

The cytochrome oxidase subunit III gene in sunflower mitochondria is cotranscribed with an open reading frame conserved in higher plants.

The gene encoding subunit III of cytochrome oxidase (COXIII) has been identified in the sunflower mitochondrial genome. The COXIII coding region is located 570 bp downstream of a 477 bp open reading frame (ORFB). Sequence comparisons and hybridization experiments show that ORFB sequences are conserved in other plant mitochondrial genomes. Nucleotide and amino acid sequence comparisons suggest that RNA editing is required in sunflower mitochondria to synthesize a functional COXIII polypeptide.

Amino Acid Sequence↗

The cytochrome oxidase subunit I gene of Tetrahymena: a 57 amino acid NH2-terminal extension and a 108 amino acid insert.

The gene sequence for cytochrome oxidase subunit I (COI) in the ciliate Tetrahymena mitochondrial DNA has been determined and shown to be coded by the same strand as codes the genes (in order) for 14S rRNA, tRNA(trp), tRNA(glu), 21S rRNA, tRNA(leu) and tRNA(met). The predicted protein has 698 amino acids, including an NH2-terminal 57 amino acid extension and a 108 amino acid insert originally found in Paramecium COI. These extension and insert segments are not highly hydrophobic but are relatively rich in lysine, arginine and serine. In analogy with the presequence of nuclear-encoded mitochondrial proteins, they might function as a transmembrane signal. The remaining polypeptide segments show a hydrophobicity characteristic of membrane spanning proteins. TCOI shows a 64% amino acid identity with Paramecium COI but less than a 38% amino acid conservation with human COI. The Tetrahymena mitochondrial code is analogous with the mammalian mitochondrial code; but differs from the Tetrahymena nuclear genetic code; TGA is exclusively translated as tryptophan; ATA is used as an initiation codon probably for methionine, and TAA as a stop codon; the arginine codons (CGN) are not used. The use of the leucine codon TTA in TCOI is contradictory to the codon recognition pattern previously obtained from the isolated tRNA(leu) isoacceptors recognizing only the CUN codons, but consistent with the tRNA(leu) (anticodon UAA) gene encoded in the genome. The reason for this inconsistency has not been resolved.

Amino Acid Sequence↗

Effect of aging and acetyl-L-carnitine on the activity of cytochrome oxidase and adenine nucleotide translocase in rat heart mitochondria.

The effect of aging and treatment with acetyl-L-carnitine on the activity of cytochrome oxidase and adenine nucleotide translocase in rat heart mitochondria was studied. It was found that the activity of both these mitochondrial protein systems was reduced (by around 30%) in aged animals. Treatment of aged rats with acetyl-L-carnitine almost completely reversed this effect. Changes in the mitochondrial cardiolipin content appear to be responsible for these effects of acetyl-L-carnitine.

Acetylcarnitine↗

Development of cytochrome oxidase staining in the retina and lateral geniculate nucleus: a possible correlate of ON- and OFF-center channel maturation.

A histochemical stain for cytochrome oxidase (CO) activity was used to examine the maturation of a neurochemical correlate of ON and OFF channels in the retina and dorsal lateral geniculate nucleus (LGN) of the tree shrew. In the adult tree shrew, the CO staining pattern can be used as a histochemical marker of segregated ON- and OFF-center channels in the retina, LGN, and striate cortex. Our previous studies have shown that the retina is immature and the LGN unlaminated at birth. In the present study, we show that the laminar development of CO reactivity emerges during the first postnatal week in the LGN, while the maturation of CO staining in the presumed ON and OFF sublaminae of the retinal inner plexiform layer develops slowly, well after the appearance of differential laminar CO staining in the LGN.

Animals↗

Magainin oligomers reversibly dissipate delta microH+ in cytochrome oxidase liposomes.

Magainin peptides present in the skin of Xenopus laevis and identified as antimicrobial agents are shown to decrease the membrane potential in cytochrome oxidase liposomes. They also released respiratory control with a third or higher order concentration dependence. Respiratory control was restored by proteolytic digestion of the added magainin. The amount of magainin required for half-maximal stimulation of respiration was proportional to lipid concentration. At appreciably higher concentrations magainins inhibited uncoupled respiration. The results are discussed in terms of a model in which most of the added magainin adsorbs as a monomer to the membranes but equilibrates with a multimeric pore that causes rather general permeability of membranes. The ensuing ion permeation dissipates membrane potential and stimulates respiration.

Animals↗

[Structural and functional characterization of cytochrome oxidase encapsulated in liposomes in sodium chloride solutions].

The effect of NaCl concentration (0-2 M) on the absorption spectrum and enzymic activity of cytochrome oxidase built in lecithin liposome has been studied at 24 and 8 degrees C. It is shown that at 0,8 M NaCl concentration a conformational transition occurs that leads to the enzyme inactivation. The relationship between protein conformation and liposome state has been established.

Animals↗

A genomewide linkage-disequilibrium scan localizes the Saguenay-Lac-Saint-Jean cytochrome oxidase deficiency to 2p16.

Leigh syndrome (LS) affects 1/40,000 newborn infants in the worldwide population and is characterized by the presence of developmental delay and lactic acidosis and by a mean life expectancy variously estimated at 3-5 years. Saguenay-Lac-Saint-Jean (SLSJ) cytochrome oxidase (COX) deficiency (LS French-Canadian type [LSFC] [MIM 220111]), an autosomal recessive form of congenital lactic acidosis, presents with developmental delay and hypotonia. It is an LS variant that is found in a geographically isolated region of Quebec and that occurs in 1/2,178 live births. Patients with LSFC show a phenotype similar to that of patients with LS, but the two groups differ in clinical presentation. We studied DNA samples from 14 patients with LSFC and from their parents, representing a total of 13 families. Because of founder effects in the SLSJ region, considerable linkage disequilibrium (LD) was expected to surround the LSFC mutation. We therefore performed a genomewide screen for LD, using 290 autosomal microsatellite markers. A single marker, D2S1356, located on 2p16, showed significant (P < 10(-5)) genomewide LD. Using high-resolution genetic mapping with additional markers and four additional families with LSFC, we were able to identify a common ancestral haplotype and to limit the critical region to approximately 2 cM between D2S119 and D2S2174. COX7AR, a gene encoding a COX7a-related protein, had previously been mapped to this region. We determined the genomic structure and resequenced this gene in patients with LSFC and in controls but found no functional mutations. Although the LSFC gene remains to be elucidated, the present study demonstrates the feasibility of using a genomewide LD strategy to localize the critical region for a rare genetic disease in a founder population.

Base Sequence↗

A new carbon monoxide-induced complex of cytochrome c oxidase.

Cytochrome c oxidase isolated from ox heart forms a complex in the presence of millimolar concentrations of CO with absorption bands at 606, 565 and 435 nm (difference spectrum), distinct from both ferrocytochrome a and the classical 590nm carbon-monoxyferrocytochrome a3. This species, which closely resembles Compound C, the derivative formed on photolysis and oxygenation of mixed-valence cytochrome a3+a32+CO, may represent a cytochrome a32+CO complex in which the associated ('invisible') copper is still oxidized.

Carbon Monoxide↗

Cytochrome oxidase deficiency presenting as birth asphyxia.

Hypoxic-ischaemic encephalopathy (HIE) was diagnosed in an infant with acidosis. At 7 weeks of age further investigations revealed abnormal neuroimaging (CT and MRI scans) and a raised plasma and CSF lactate. A skeletal-muscle biopsy at 2 months of age confirmed the diagnosis of cytochrome oxidase deficiency. The course of the patient's disorder has taken that of a static encephalopathy (cerebral palsy). Inborn disorders of the respiratory chain should be considered in the differential diagnosis of HIE.

Acidosis, Lactic↗

A ligand-exchange mechanism of proton pumping involving tyrosine-422 of subunit I of cytochrome oxidase is ruled out.

The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system. Site-directed mutants of Y422 have been constructed in the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides to test this hypothesis (Y422A, Y422F). The results demonstrate that Y422 is not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a3, as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed electron transfer between heme a and heme a3 is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residue previously postulated to interact electrostatically with heme a3, nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.

Base Sequence↗

Subunit 1 of cytochrome oxidase from Neurospora crassa: nucleotide sequence of the coding gene and partial amino acid sequence of the protein.

A partial protein sequence (223 residues) of cytochrome oxidase subunit 1 from Neurospora crassa has been established. The nucleotide sequence of a cloned mitochondrial DNA segment, including the structural gene coding for the mature subunit 1 (CO I locus) was determined. In contrast to the situation in yeast, the CO I locus in N. crassa is not interrupted by long intervening sequences. A polypeptide of 555 residues with a mol. wt. of 61 000 has been deduced from the reading frame established by protein sequencing. With the exception of the C-terminal part of the polypeptide, the proposed sequences for subunit 1 of N. crassa, yeast, and man are largely homologous. Protein sequencing reveals that a region of low homology close to the C-terminal portion belongs to the structural gene in N. crassa. The DNA sequence coding for the prepiece , which characterizes the polypeptide precursor of the N. crassa subunit 1, has not yet been localized. A RNA species of approximately 6.8 kb has been identified as the CO I transcript. There is no indication of splicing of this large transcript.

Amino Acid Sequence↗

Phylogeny and life history evolution of the genus Chrysoritis within the Aphnaeini (Lepidoptera: Lycaenidae), inferred from mitochondrial cytochrome oxidase I sequences.

Phylogenetic relationships among 26 South African species in the tribe Aphnaeini (Lepidoptera: Lycaenidae) were inferred from DNA characters of the mitochondrial gene cytochrome oxidase I (COI), using maximum-parsimony methods. The resulting phylogenetic estimate supports the systematic hypothesis made by Heath (1997, Metamorphosis, supplement 2), based on morphological characters, that at least three preexisting genera (Chrysoritis, Poecilmitis, and Oxychaeta) should be collapsed into the single monophyletic genus Chrysoritis. Two of the species groups described by Heath within Chrysoritis are also monophyletic, while one is paraphyletic and thus unsupported by the molecular data. Strong node support and skewed transition/transversion ratios suggest that two Chrysoritis clades contain synonymous species. Aphytophagy appears as a derived feeding strategy. Evolutionary patterns of ant association indicate lability at the level of ant genus, while association with different ant subfamilies may have played an ancestral and chemically mediated role in the diversification of South African aphnaeines.

Animals↗

The cytochrome oxidase II gene in mitochondria of the sugar-beet Beta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologous coxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89-95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beet coxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plant coxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestral coxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beet coxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons of coxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon of coxII was identified in the mtDNA of the sugar-beet. This 'pseudo-gene' is transcribed and its existence in the mitochondrial genome is unexplained.

Amino Acid Sequence↗