Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.     

Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.

We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.     

Identification by RNA-based arbitrarily primed PCR of the involvement of cytochrome c oxidase in the development of resistance to methotrexate

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.     

Mitochondrial cytochrome C oxidase subunit I of Manduca sexta and a comparison with other invertebrate genes

A cDNA encoding mitochondrial cytochrome c oxidase subunit I (mt COI) from Manduca sexta (Lepidoptera: Sphingidae) was cloned and sequenced. AT (adenine-thymine) content is high and codon usage is biased and likely reflects the role of mt COI in electron transport. The encoded protein is 514 amino acids long, contains seven invariant His residues observed in COIs in all organisms and would be predicted to be composed of 12 transmembrane regions.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

The occurrence of beta-hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens

The preliminary data on the amino acid sequence of subunit IV from bovine heart cytochrome oxidase (Albany) is presented. The subunit consists of 97 amino acids linked together in a single polypeptide chain. The sequence was established by the isolation, purification and sequencing of some of the tryptic, chymotryptic and thermolytic and Staphylococcus aureus protease peptides. This subunit is present in all cytochrome oxidase preparations. It corresponds to polypeptide VIa in cytochrome oxidase (Aachen) and subunit a in cytochrome oxidase (Eugene).     

A molecular phylogenetic study of the freshwater talitrid amphipod Orchestia cavimana (Crustacea).

In this paper we performed a molecular phylogenetic study of Orchestia cavimana, the sole talitrid amphipod inhabiting beaches of European freshwater lakes and rivers. For that purpose, we have PCR amplified and sequenced regions of the mitochondrial cytochrome oxidase subunit I (COI) gene, basing our analysis on both nucleotide and amino acid sequences and considering also structural classes of the COI enzyme. Phylogenetic analyses were conducted by neighbour-joining (NJ) and maximum-parsimony (MP) methods comparing homologous sequences of talitrids and other Crustacea. In both NJ and MP trees, O. cavimana shows a basal placement with respect to other talitrid amphipods.     

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.     

Molecular cloning of the cytochrome aa3 gene from the archaeon (Archaebacterium) Halobacterium halobium.

A novel aa3-type cytochrome oxidase from the extremely halophilic archaeon, Halobacterium halobium, differs significantly from those of other prokaryotic and eukaryotic cytochrome oxidases (Fujiwara, T., Fukumori, Y., and Yamanaka, T. (1989) J. Biochem. 105, 287-292). In the present study, we cloned and sequenced the gene which encodes the cytochrome aa3 by using the polymerase chain reaction methods. The deduced amino acid sequence of subunit I of H. halobium cytochrome aa3 was more similar to that of subunit I of the eukaryotic cytochrome (44%, maize mitochondria) than that of the cytochrome from other bacteria (36%, Paracoccus denitrificans). The consensus sequence in putative metal binding residues is well-conserved also in H. halobium cytochrome aa3.     

Cytochrome aa3 in Haloferax volcanii

A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3). This cytochrome aa(3) consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.     

Amino acid sequence of subunit V of bovine heart cytochrome oxidase, the heme alpha-containing subunit.

The complete amino acid sequence of the heme alpha-containing subunit V of bovine heart cytochrome oxidase was determined to be: H2N-Ser-His-Gly-Ser-His-Glu-Thr-Asp-Glu-Glu-Phe-Asp-Ala-Arg-Trp-Val-Thr-Tyr-Phe-Asn-Lys-Pro-Asp-Ile-Asp-Ala-Trp-Glu-Leu-Arg-Lys-Gly-Met-Asn-Thr-Leu-Val-Gly-Tyr-Asp-Leu-Val-Pro-Glu-Pro-Lys-Ile-Ile-Asp-Ala-Ala-Leu-Arg-Ala-Cys-Arg-Arg-Leu-Asn-Asp-Phe-Ala-Ser-Ala-Val-Arg-Ile-Leu-Glu-Val-Val-Lys-Asp-Lys-Ala-Gly-Pro-His-Lys-Glu-Ile-Tyr-Pro-Tyr-Val-Ile-Gln-Glu-Leu-Arg-Pro-Thr-Leu-Asn-Glu-Leu-Gly-Ile-Ser-Thr-Pro-Glu-Glu-Leu-Gly-Leu-Asp-Lys-Val-COOH. The subunit V is a single polypeptide which consists of 109 amino acid residues. The protein contains 48.6% hydrophobic residues and 34.0% hydrophilic residues and it is an acidic protein having a net charge of -3 at neutral pH. The molecular weight of subunit V was calculated to be 12,436 and that for the heme alpha-containing polypeptide was 13,295.     

Mitochondrial COI sequences of brachiopods: genetic code shared with protostomes and limits of utility for phylogenetic reconstruction

A 1230-bp region of the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA of each of 16 brachiopod species, representing all five living orders, was amplified by polymerase chain reaction and sequenced. Pairwise comparisons of sequence differences plotted against divergence times estimated from the brachiopod fossil record revealed that, although there are considerable variations in the expected substitution rate among different lineages, amino acid substitutions of the COI sequences may largely become saturated in 100 Ma, due mostly to multiple substitutions at the same site. Coinciding with this result, phylogenetic analysis indicated low bootstrap values for nodes corresponding to divergence events that occurred before 100 Ma, suggesting that COI sequences are suitable only for inference of phylogenetic events subsequent to the Mesozoic. Examination of brachiopod codons corresponding to invariant amino acids in the COI of various other animals suggest the nonuniversal codon relationships UGA = Trp, AUA = Met, AAA/G = Lys, and AGA/G = Ser. These are identical to those in mollusks, annelids, and arthropods, consistent with the conclusion that brachiopods are protostomes, as indicated by previous molecular analyses.     

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase in inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II-IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site of subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.     

Chicken mitochondrial cytochrome C oxidase subunit II: comparative analysis among the vertebrates

The chicken cytochrome c oxidase subunit II (COII) was cloned and sequenced. A comparison of the deduced chicken COII sequence with 4 other vertebrate counterparts revealed 64-66% amino acid sequence homology and 68-70% nucleotide sequence homology. Four peptide segments each of nine amino acids long are highly conserved across the 5 species. A redox-center was formed by three of these highly conserved domains, which include two invariant Cys and two invariant His residues for copper ion coordination, three strictly conserved Glu or Asp residues for cytochrome c binding, and highly conserved aromatic acid residues for electron transfer.     

Mitochondrial DNA sequence variation in the spruce budworm species complex (Choristoneura: Lepidoptera)

A combination of polymerase-chain-reaction amplification and automated DNA sequencing was used to survey variation in a species complex of pest insects, the spruce budworms (Choristoneura fumiferana species group), and an outgroup species, C. rosaceana. We sequenced an mtDNA region of 1,573 bp that extends from the middle of cytochrome oxidase subunit I (COI) through tRNA leucine (UUR) to the end of cytochrome oxidase subunit II. In addition, we examined levels of intraspecific variation within a 470-bp region of the COI gene. Choristoneura fumiferana clearly represented the oldest lineage within its species group, with 2.7%-2.9% sequence divergence from the other species. In contrast, the four remaining species (C. pinus, C. biennis, C. occidentalis, and C. orae) had closely related or identical mtDNA, with < 1% divergence among most of their haplotypes. Despite its older lineage and widespread geographic distribution, C. fumiferana showed significantly lower intraspecific genetic diversity than did C. occidentalis. Choristoneura orae shared haplotypes with C. occidentalis and C. biennis, and species-level separation of these three species was not supported. Two divergent, uncommon haplotypes were also found in C. occidentalis and C. biennis. The divergent haplotype in C. biennis had an unusually high number of inferred amino acid replacements, suggesting selective differences between mitochondrial DNA haplotypes. Transition:transversion ratios in Choristoneura paralleled those found in Drosophila; transition:transversion ratios were highest in closely related sequences but decreased with increasing sequence divergence. Nucleotide composition showed an A+T bias that was near the high end of the range known for insects. This work illustrates the potential utility of direct DNA sequencing in assessing population structures, species limits, and phylogenetic relationships among organisms that have not previously been subjected to DNA analysis.      

The most conserved nuclear-encoded polypeptide of cytochrome c oxidase is the putative zinc-binding subunit: primary structure of subunit V from the slime mold Dictyostelium discoideum.

A full-length 515 base pairs cDNA for cytochrome c oxidase subunit V of D. discoideum was isolated from a lambda gt11 expression library. The encoded polypeptide, whose identity was confirmed by partial protein sequencing, is 119 amino acids long (Mr = 13,352) and does not contain a cleavable presequence. The protein, which is homologous to human subunit Vb and yeast subunit IV, exhibits the highest degree of sequence conservation found among nuclear-encoded subunits of cytochrome c oxidase from distantly related organisms. All the invariant residues are clustered in two regions of the C-terminus which include the putative amino acids involved in the coordination of the Zn ion tightly associated to eukaryotic oxidase.     

A processed pseudogene with an intact coding sequence for rat liver cytochrome c oxidase subunit VIc

The biogenesis of eukaryotic cytochrome c oxidase involves the coordinate expression of nuclear and mitochondrial genes. Very little information is available on the gene structure of nuclear-coded cytochrome c oxidase subunits in mammalian systems. We report here the isolation and complete nucleotide sequence determination of a processed pseudogene for cytochrome c oxidase subunit VIc from rat liver. The pseudogene lacks introns and the coding region is intact with no deleterious lesions; however, there are 7 amino acid (aa) differences when compared to the sequence derived from cDNA clones. The pseudogene has the potential to code for a protein of 76 aa, containing a putative 3 aa N-terminal presequence when compared to the mature bovine heart VIc subunit. Potential regulatory regions, including a TATA box, are present in the 5'-flanking region.