Molecular Evolution of Cytochrome c Oxidase Subunit IV: Evidence for Positive Selection in Simian Primates

Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of ``neutral DNA' nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages. Received: 22 May 1996 / Accepted: 24 November 1996     

The accelerated evolution of human cytochrome c oxidase – Selection for reduced rate and proton pumping efficiency?

The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.     

Evolutionary Rate Acceleration of Cytochrome c Oxidase Subunit I in Simian Primates

We present an analysis of the evolutionary rates of the cytochrome c oxidase subunit I genes of primates and other mammals. Five primate genes were sequenced, and this information was combined with published data from other species. The sequences from simian primates show approximately twofold increases in their nonsynonymous substitution rate compared to those from other primates and other mammals. The species range and the overall magnitude of this rate increase are similar to those previously identified for the cytochrome c oxidase subunit II and cytochrome b genes. Received: 22 July 1999 / Accepted: 21 February 2000     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Molecular evolution of the cytochrome c oxidase subunit <Emphasis Type="Italic">5</Emphasis> A gene in primates

Background  

Many electron transport chain (ETC) genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains.     

Accelerated Evolution of Cytochrome b in Simian Primates: Adaptive Evolution in Concert with Other Mitochondrial Proteins?

We have sequenced the cytochrome b gene of Horsfield's tarsier, Tarsius bancanus, to complete a data set of sequences for this gene from representatives of each primate infraorder. These primate cytochrome b sequences were combined with those from representatives of three other mammalian orders (cat, whale, and rat) in an analysis of relative evolutionary rates. The nonsynonymous nucleotide substitution rate of the cytochrome b gene has increased approximately twofold along lineages leading to simian primates compared to that of the tarsier and other primate and nonprimate mammalian species. However, the rate of transversional substitutions at fourfold degenerate sites has remained uniform among all lineages. This increase in the evolutionary rate of cytochrome b is similar in character and magnitude to that described previously for the cytochrome c oxidase subunit II gene. We propose that the evolutionary rate increase observed for cytochrome b and cytochrome c oxidase subunit II may underlie an episode of coadaptive evolution of these two proteins in the mitochondria of simian primates. Received: 15 December 1997 / Accepted: 24 February 1998     

Molecular evolution of aerobic energy metabolism in primates

As part of our goal to reconstruct human evolution at the DNA level, we have been examining changes in the biochemical machinery for aerobic energy metabolism. We find that protein subunits of two of the electron transfer complexes, complex III and complex IV, and cytochrome c, the protein carrier that connects them, have all undergone a period of rapid protein evolution in the anthropoid lineage that ultimately led to humans. Indeed, subunit IV of cytochrome c oxidase (COX; complex IV) provides one of the best examples of positively selected changes of any protein studied. The rate of subunit IV evolution accelerated in our catarrhine ancestors in the period between 40 to 18 million years ago and then decelerated in the descendant hominid lineages, a pattern of rate changes indicative of positive selection of adaptive changes followed by purifying selection acting against further changes. Besides clear evidence that adaptive evolution occurred for cytochrome c and subunits of complexes III (e.g., cytochrome c(1)) and IV (e.g., COX2 and COX4), modest rate accelerations in the lineage that led to humans are seen for other subunits of both complexes. In addition the contractile muscle-specific isoform of COX subunit VIII became a pseudogene in an anthropoid ancestor of humans but appears to be a functional gene in the nonanthropoid primates. These changes in the aerobic energy complexes coincide with the expansion of the energy-dependent neocortex during the emergence of the higher primates. Discovering the biochemical adaptations suggested by molecular evolutionary analysis will be an exciting challenge.     

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.     

Molecular Evolution at the Cytochrome Oxidase Subunit 2 Gene Among Divergent Populations of the Intertidal Copepod, Tigriopus californicus

The cytochrome c oxidase subunit 2 gene (COII) encodes a highly conserved protein that is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX) crucial to the production of ATP during cellular respiration. Despite its integral role in electron transport, we have observed extensive intraspecific nucleotide and amino acid variation among 26 full-length COII sequences sampled from seven populations of the marine copepod, Tigriopus californicus. Although intrapopulation divergence was virtually nonexistent, interpopulation divergence at the COII locus was nearly 20% at the nucleotide level, including 38 nonsynonymous substitutions. Given the high degree of interaction between the cytochrome c oxidase subunit 2 protein (COX2) and the nuclear-encoded subunits of COX and cytochrome c (CYC), we hypothesized that some codons in the COII gene are likely to be under positive selection in order to compensate for amino acid substitutions in other subunits. Estimates of the ratio of nonsynonymous to synonymous substitution (ω), obtained using a series of maximum likelihood models of codon substitution, indicated that the majority of codons in T. californicus COII are under strong purifying selection (ω << 1), while approximately 4% of the sites in this gene appear to evolve under relaxed selective constraint (ω = 1). A branch-site maximum likelihood model identified three sites that may have experienced positive selection within the central California sequence clade in our COII phylogeny; these results are consistent with previous studies showing functional and fitness consequences among interpopulation hybrids between central and northern California populations. [Reviewing Editor: Dr. Willie Swanson]     

Adaptive evolution of the <Emphasis Type="Italic">Homo</Emphasis> mitochondrial genome

Adaptive evolution of 12 protein-coding mitochondrial genes in members of genus Homo (Denisova hominin (H. sp. Altai), Neanderthals (H. neanderthalensis) and modern humans (H. sapiens)) has been evaluated by assessing the pattern of changes in the physicochemical properties of amino acid replacements during primate evolution. It has been found that molecular adaptation (positive destabilizing selection) in Homo becomes apparent in the form of 12 radical amino acid replacements accompanied with statistically significant (P < 0.001) changes of physicochemical properties that probably had functional consequences. These replacements occurred at the stage of a common ancestor of Homo (in CO2 and CytB genes) as well as with the appearance of the common ancestor of Neanderthals and modern humans (in CO1 and ND5 genes). Radical amino acid replacements were mainly revealed in the cytochrome c oxidase complex IV and cytochrome bc1 complex III, thus coinciding with the general trend of increasing nonsynonymous changes in mtDNA genes coding subunits of complexes’ III and IV proteins in anthropoid primates.     

Evolution of the primate cytochrome c oxidase subunit II gene

We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. Correspondence to: R.L. Honeycutt     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

Physical and functional characterisation of monomeric and dimeric eukaryotic cytochrome c oxidases

Cytochrome c oxidases were isolated from heart tissue of beef (Bos tauros), sheep (Ovis aries), horse (Equus caballus), pig (Sus scrofa) (native dimers) and hammerhead shark (Sphyrna lewinii) (native monomer). Limited proteolysis of dimeric enzymes selectively depleted subunit III, resulting in monomerisation and a blue shift (2nm) of the reduced α band to the same wavelength maximum (603nm) as that of the hammerhead shark enzyme. Monomeric enzymes retain the ability to accept electrons rapidly from cytochrome c, and the second-order rate constants for electron transfer between cytochromes c and a are reported. The steady-state kinetics of both native and subunit III-depleted cytochrome c oxidases were biphasic, thus ruling out any explanation for this behaviour that depends on cooperation between functional units (monomers) within a dimer. Functional integrity of the subunit III-depleted enzyme prepared by proteolysis was maintained during multiple turnover, in contrast to reports elsewhere of loss of activity caused by subunit III removal by other means. A model is proposed to explain this difference, in which removal of a hydrophobic membrane-spanning segment of subunit III leads to monomerisation but a residual extra-membrane segment is retained, preserving the functional integrity of the enzyme.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

Molecular evolution of cytochrome c oxidase subunit I in primates: is there coevolution between mitochondrial and nuclear genomes?

Phylogenetic analyses carried out on cytochrome c oxidase (COX) subunit I mitochondrial genes from 14 primates representing the major branches of the order and four outgroup nonprimate eutherians revealed that transversions and amino acid replacements (i.e., the more slowly occurring sequence changes) contained lower levels of homoplasy and thus provided more accurate information on cladistic relationships than transitions (i.e., the more rapidly occurring sequence changes). Several amino acids, each with a high likelihood of functionality involving the binding of cytochrome c or interaction with COX VIII, have changed in Anthropoidea, the primate suborder grouping New World monkey, Old World monkey, ape, and human lineages. They are conserved in other mammalian lineages and in nonanthropoid primates. Maximum-likelihood ancestral COX I nucleotide sequences were determined utilizing a near most parsimonious branching arrangement for the primate sequences that was consistent with previously hypothesized primate cladistic relationships based on larger and more diverse data sets. Relative rate tests of COX I mitochondrial sequences showed an elevated nonsynonymous (N) substitution rate for anthropoid-nonanthropoid comparisons. This finding for the largest mitochondrial (mt) DNA-encoded subunit is consistent with previous observations of elevated nonsynonymous substitution/synonymous substitution (S) rates in primates for mt-encoded COX II and for the nuclear-encoded COX IV and COX VIIa-H. Other COX-related proteins, including cytochrome c and cytochrome b, also show elevated amino acid replacement rates or N/S during similar time frames, suggesting that this group of interacting genes is likely to have coevolved during primate evolution.     

Structure and evolution of opossum,guinea pig,and porcupine cytochrome b genes

Summary We have sequenced the mitochondrial cytochrome b gene from the guinea pig, the African porcupine, and a South American opossum. A phylogenetic analysis, which includes 22 eutherian and four other vertebrate cytochrome b sequences, indicates that the guinea pig and the porcupine constitute a natural clade (Hystricomorpha) that is not a sister group to the clade of mice and rats (Myomorpha). Therefore, the hypothesis that the Rodentia is paraphyletic receives additional support. The artiodactyls, the perissodactyls, and the cetaceans form a group that is separated from the primates and the rodents. The 26 sequences are used to study the structure/function relationships in cytochrome b, whose function is electron transport. Most of the amino acid residues involved in the two reaction centers are well conserved in evolution. The four histidines that are believed to ligate the two hemes are invariant among the 26 sequences, but their nearby residues are not well conserved in evolution. The eight transmembrane domains represent some of the most divergent regions in the cytochrome b sequence. The rate of nonsynonymous substitution is considerably faster in the human and elephant lineages than in other eutherian lineages; the faster rate might be due to coevolution between cytochrome b and cytochrome c. Offprint requests to: W.-H. Li     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.     

Mutations in CYC1, Encoding Cytochrome c1 Subunit of Respiratory Chain Complex III,Cause Insulin-Responsive Hyperglycemia

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c₁ subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c₁, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c₁ is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals’ fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c₁ stability and complex III activity.     

Protein–protein interactions of the cytochrome c oxidase DNA barcoding region

Enzymatic amplification of homologous regions of DNA using ‘universal’ polymerase chain reaction primers has provided insight into insect systematics, phylogeography, molecular evolution and species identification. One of the more commonly amplified and sequenced regions is a short region of the cytochrome c oxidase subunit I gene (COI), commonly called the barcoding region. COI is one of three mitochondrial‐encoded subunits of complex IV (Cox) of the electron transport chain. In addition to the mitochondrial subunits there are nine nuclear‐encoded subunits of the complex in Drosophila. Whereas a number of phylogenetic biases associated with this region have been examined and the quaternary structure of Cox has been modelled, the influence of protein–protein interactions on the observed patterns of evolution in this barcoding region of insects has never been examined critically. Using a well‐resolved independently derived phylogeny of 38 Diptera species, we examined the homogeneity of the substitution processes within the barcoding region. We show that, within Diptera, amino acid residues interacting with nuclear‐encoded subunits of Cox are evolving at elevated rates across the phylogeny. Furthermore, we show that codon position two is biased by protein–protein interactions. In contrast, third codon positions provide a less biased estimate of genetic variation in the region. This study highlights the need to examine the potential for systematic bias in DNA barcoding regions as part of the critical assessment of evidence in systematics and in biodiversity assessments.     

Exceptional longevity and exceptionally high metabolic rates in anthropoid primates are linked to a major modification of the ubiquinone reduction site of cytochrome b

The maximal lifespan of Anthropoid primates (monkeys, apes and humans) exceed the lifespan of most other mammals of equal body mass. Unexpectedly, their exceptional longevity is associated with exceptionally high metabolic rates, in apparent contradiction to the Free Radical Theory of Aging. It was therefore suggested that in anthropoid primates (and several other taxa of mammals and birds) the mitochondrial electron transport complexes evolved to modify the relationship between basal electron transport and superoxide generation to allow for the evolution of exceptional longevity. Cytochrome b, the core protein of the bc1 complex is a major source of superoxide. The amino-acid sequence of cytochrome b evolved much faster in anthropoid than in prosimian primates, and most other mammals, resulting in a large change in the amino-acids composition of the protein. As a result of these changes cytochrome b in anthropoid primates is significantly less hydrophobic and contains more polar residues than other primates and most other mammals. Most of these changes are clustered around the reduction site of uboiquinone. In particular a key positively charged residue, arginine 313, that interacts with propionate D of heme b_H, and thus raises its redox potential, is substituted in anthropoid primates with the neutral residue glutamine, most likely resulting in a lower redox potential of heme b_H and faster reduction of ubiquinone at high proton motive force. It is suggested that these changes contribute to the observed increased rates of basal metabolism and reduce the rates of superoxide production, thus allowing for increased lifespan.