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We examined inferred amino acid replacements for 16 genes that encode the proteins of the cytochrome c oxidase (COX) holoenzyme in eight vertebrate species. Phylogeny-based analysis revealed that the human lineage (primates) has had an unusually large, statistically significant, number of amino acid replacements in the mature protein coding region of these genes. This finding is similar to earlier observations of an accelerated non-synonymous substitution rate for some lineages of primates for COX1, COX2, COX4, and COX7AH. In contrast, the mitochondrial targeting presequences of these same proteins have not undergone a concomitant rate change. This more comprehensive analysis suggests that COX5A, COX6B, COX6C, COX7C, and COX8L have also undergone an acceleration in amino acid replacement rates in anthropoid primates. Some of these rate accelerations (e.g. in COX5A and COX7C) are so pronounced that non-human mammalian sequences are more similar to sequences from Xenopus or zebrafish than they are to human. Since the functions of the targeting and mature proteins of these polypeptides are different, the mature portions of these genes are likely to have undergone a functionally significant change that is adaptive in nature.

Subunit VIIa of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) exists in at least two isoforms, one present in all tissue types (‘liver’ isoform; COX VIIa-L) and the other specific for cardiac and skeletal muscle (COX VIIa-M). We have isolated a full-length cDNA encoding human COX VIIa-M. The deduced polypeptide represents the human ortholog of COX VIIa-M, as it shares 78% identity with bovine COX VIIa-M, but only 63% identity with human COX VIIa-L. Northern-blot analysis of primate tissues demonstrated that COXVIIa-M mRNA is present only in muscle tissues; in contrast, the COXVIIa-L mRNA is present in both muscle and nonmuscle tissues. Southern-blot hybridization of human-rodent cell hybrid genomic DNA indicates that the COXVHa-M gene maps to a single locus on chromosome 19, designated COX7AM. In contrast, COXVIIa-L cDNA probes hybridized to fragments from two COX7AL loci, on chromosomes 4 and 14.

We have isolated a chimpanzee processed pseudogene for subunit IV of cytochrome c oxidase (COX; EC 1.9.3.1) by screening a chimpanzee genomic library in λCharon 32 with a bovine liver cDNA encoding COX subunit IV (COX IV), and localized it to a 1.9-kb Hin dIII fragment. Southern-blot analysis of genomic DNA from five primates showed that DNAs from human, gorilla, and chimpanzee each contained the 1.9-kb pseudogene fragment, whereas orangutan and pigtail macaque monkey DNA did not. This result clearly indicates that the pseudogene arose before the divergence of the chimpanzee and gorilla from the primate lineage. By screening Chinese hamster × human hybrid panels with the human COX4 cDNA, we have mapped COX4 genes to two human chromosomes, 14 and 16. The 1.9-kb Hin dIII fragment containing the pseudogene, COX4P1 , can be assigned to chromosome 14, and by means of rearranged chromosomes in somatic cell hybrids, to 14q21-qter. Similarly, the functional gene, COX4 , has been mapped to 16q22-qter.

The terminal enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX), contains three mitochondrial and ten nuclear encoded subunits in mammals. Three of the nuclear subunits (VIa, VIIa, and VIII) have muscle and non-muscle-specific isoforms, subunit IV contains a lung-specific isoform, and subunit VIb contains a testes-specific isoform. For subunit VIII, the smallest nuclear encoded COX polypeptide, we have now found a third gene (COX 8-3), which has been identified in human, lemur, rat, and mouse, suggesting that it is present in a broad range of Eutherian mammals. Sequence similarity and gene structure support the homology of COX8-3 to the other subunit VIII isoforms, indicating that all three are the product of gene duplications. COX VIII-3 protein is mitochondrially-targeted, as shown by a fluorescent COX VIII3/DsRed fusion protein. Both the mitochondrial targeting and its sequence conservation suggest that COXVIII-3 functions as part of the COX holoenzyme and could have a tissue-specific role, as is the case for the other two isoforms. Questions remain about where COX8-3 is predominantly expressed. However, detection of full-length cDNAs, lower levels of sequence divergence at the first and second codon positions compared to the third, and a conserved gene structure indicate that COX VIII-3 is an expressed gene whose origin dates to at least 91 million years ago.

We have isolated a cDNA that encodes subunit VIIc of bovine cytochrome c oxidase (COX VIIc). The 325-bp cDNA contains sequences encoding the mature 47-amino acid (aa) polypeptide and a 16-aa presequence. The deduced aa sequence of the processed polypeptide is identical to that of the heart protein determined by aa sequencing. Northern-blot analysis reveals a single 525-nucleotide (nt) transcript in all tissues examined, whose levels vary with the corresponding respiratory activities in different tissues; thus, no evidence for isoforms of COX VIIc is seen in adult tissues. Southern-blot analysis of bovine genomic DNA digested with three different restriction enzymes reveals several bands that hybridize with the cDNA. We present here the sequence of one genomic region that contains a processed gene encoding COX VIIc. The genomic and cDNA nt sequences are 99% identical throughout the 189-bp open reading frame; the deduced aa sequences are identical. The sequence of the genomic clone suggests that the cDNA terminates prematurely at an EcoRI site in the 3'-untranslated region. We have compared COXVIIc cDNAs from cow, human and mouse, and find the presequence similarity among them to be 100% at the aa level.

We have isolated and analyzed 17 clones from a bovine genomic library in phage λ Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.