Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes

The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.     

Subunit III of cytochrome c oxidase is expressed in Paracoccus denitrificans

Polyclonal antibodies have been obtained against a synthetic dodecapeptide identical to the aminoacid sequence 120-131 DSPIKDGVWPPE (inferred from its DNA sequence) of Paracoccus denitrificans cytochrome c oxidase subunit III. The antibodies had a titer higher than 1:10000 when tested against the antigen. These antibodies have been used to produce immunological evidence that, despite the fact that subunit III is not isolated with cytochrome c oxidase, it exists in Paracoccus denitrificans lysates. The antibodies did not show reactivity with bovine heart cytochrome c oxidase either by ELISA or immunoblotting. It was also shown that the antibodies react with a single polypeptide present in Paracoccus denitrificans cell lysates, having an apparent molecular weight close to that of subunit III of bovine heart oxidase.     

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.     

DNA barcoding of Oryx leucoryx using the mitochondrial cytochrome C oxidase gene

The massive destruction and deterioration of the habitat of Oryx leucoryx and illegal hunting have decimated Oryx populations significantly, and now these animals are almost extinct in the wild. Molecular analyses can significantly contribute to captive breeding and reintroduction strategies for the conservation of this endangered animal. A representative 32 identical sequences used for species identification through BOLD and GenBank/NCBI showed maximum homology 96.06% with O. dammah, which is a species of Oryx from Northern Africa, the next closest species 94.33% was O. gazella, the African antelope. DNA barcode sequences of the mitochondrial cytochrome C oxidase (COI) gene were determined for O. leucoryx; identification through BOLD could only recognize the genus correctly, whereas the species could not be identified. This was due to a lack of sequence data for O. leucoryx on BOLD. Similarly, BLAST analysis of the NCBI data base also revealed no COI sequence data for the genus Oryx.     

A gene in Paracoccus for subunit III of cytochrome oxidase

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.     

Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects.

We examine the complete nucleotide sequences of the mitochondrial cytochrome oxidase II gene of 13 species of insects, representing 10 orders. The genes range from 673 to 690 bp in length, encoding 226 to 229 amino acids. Several insertion or deletion events, each involving one or two codons, can be observed. The 3' end of the gene is extremely variable in both length and sequence, making alignment of the ends unreliable. Using the first 639 nucleotide positions, for which unambiguous alignments could be obtained, we examine the neighbor-joining trees based on nucleotide divergences and based on conserved subsets of that data, including transversion and amino acid and second codon position divergences. Each of these subsets produces different trees, none of which can be easily reconciled with trees constructed using morphology and the fossil record. Bootstrap analysis using second codon positions strongly supports affinities between the order Blatteria (cockroaches) and the order Isoptera (termites) and between a wasp and the published honeybee sequence (Order Hymenoptera). The divergence of insect orders is very ancient and may have occurred too rapidly for easy resolution using mitochondrial protein sequences. Unambiguous resolution of insect orders will probably require analysis of many additional taxa, using the COII gene and other conserved sequences.     

Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.     

Effect of oxygen on the synthesis and assembly of mitochondrial encoded subunits of cytochrome oxidase and cytochrome b.c1 in mouse embryo fibroblasts.

Mouse embryo fibroblasts were grown in low and control O2 for 24 h (average medium oxygen tensions, 7 torr and 143 torr, respectively). Relative to controls, there was a reduction in radiolabeled subunits in immunoprecipitates of cytochrome oxidase and cytochrome b.c1 prepared from low O2 cells. Incorporation of radiolabeled amino acids into subunit I of cytochrome oxidase and the apocytochrome b protein of the b.c1 complex ranged from 51-100% of control, whereas the appearance of these pulse-labeled subunits into holoenzymes immunoprecipitated from low O2 cells was in the range of 6-39% of control. The synthesis of subunit II of cytochrome oxidase by low O2 cells ranged from 63-100% of control, and assembly of this protein into the low O2 immunoprecipitated enzyme ranged from 15-61% of control. Thus, the data suggest that O2 had an effect on the assembly of these mitochondrially translated proteins that was independent of any effect on their synthesis.     

Species-specific identification of human hookworms by PCR of the mitochondrial cytochrome oxidase I gene.

Significant differences in the life histories of the human hookworms Ancylostoma duodenale and Necator americanus necessitate their differentiation for epidemiological studies and the design of control programs. Current methods of identification require time-consuming, labor-intensive techniques. A polymerase chain reaction (PCR)-based method that enables rapid species identification is described. The mitochondrial cytochrome oxidase I genes of both species were sequenced, and species-specific primer sets were designed. The primers were used in PCR to amplify 585-bp fragments of the cytochrome oxidase gene from individual hookworm eggs, larvae, and adults. The technique was also able to identify mixed infections containing equal amounts of eggs from each species. The technique is rapid, technically simple, and sensitive and will permit the accurate identification of human hookworms in epidemiological field studies.