Nuclear-encoded subunits of human cytochrome c oxidase: SstI restriction fragment length polymorphism

A DNA polymorphism of the nuclear-encoded subunit Va of the human cytochrome c oxidase (COX), a mitochondrial respiratory enzyme, is reported. No polymorphism was detected in genes for the subunits IV and Vb of the same enzyme.     

Investigating the role of the physiological isoform switch of cytochrome c oxidase subunits in reversible mitochondrial disease

Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues.We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Cytochrome c oxidase deficiency: Patients and animal models

Cytochrome c oxidase (COX) deficiencies are one of the most common defects of the respiratory chain found in mitochondrial diseases. COX is a multimeric inner mitochondrial membrane enzyme formed by subunits encoded by both the nuclear and the mitochondrial genome. COX biosynthesis requires numerous assembly factors that do not form part of the final complex but participate in prosthetic group synthesis and metal delivery in addition to membrane insertion and maturation of COX subunits. Human diseases associated with COX deficiency including encephalomyopathies, Leigh syndrome, hypertrophic cardiomyopathies, and fatal lactic acidosis are caused by mutations in COX subunits or assembly factors. In the last decade, numerous animal models have been created to understand the pathophysiology of COX deficiencies and the function of assembly factors. These animal models, ranging from invertebrates to mammals, in most cases mimic the pathological features of the human diseases.     

CYTOCHROME C OXIDASE ACTIVITY IN INTERPOPULATION HYBRIDS OF A MARINE COPEPOD: A TEST FOR NUCLEAR-NUCLEAR OR NUCLEAR-CYTOPLASMIC COADAPTATION

The respiratory enzyme cytochrome c oxidase (COX) is composed of subunits encoded by both nuclear and mitochondrial genes; thus, COX activity reflects, to some extent, the coordinated function of the two genomes. Because extensive mtDNA differentiation exists between populations of the copepod Tigriopus californicus, we hypothesized that laboratory hybridizations that disrupt natural combinations of nuclear and mitochondrial genes might negatively impact COX activity. Although experimental results varied greatly among different crosses, replicate sets of crosses between two particular populations showed consistent evidence for nuclear-cytoplasmic coadaptation.     

A Candidate Complex Approach to Study Functional Mitochondrial DNA Changes: Sequence Variation and Quaternary Structure Modeling of Drosophila simulans Cytochrome c Oxidase

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Oxa1-Ribosome Complexes Coordinate the Assembly of Cytochrome c Oxidase in Mitochondria

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9–10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

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     

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]     

Cytochromec oxidase ofEuglena gracilis: Purification,characterization, and identification of mitochondrially synthesized subunits

Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [³⁵S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec ₅₅₈ and has monophasic kinetics. Using horse cytochromec ₅₅₀ as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec ₅₅₀ and 35-fold higher with theEuglena cytochromec ₅₅₈. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number     

Rapid Nonsynonymous Evolution of the Iron-Sulfur Protein in Anthropoid Primates

Cytochrome c (CYC) and 9 of the 13 subunits of cytochrome c oxidase (complex IV; COX) were previously shown to have accelerated rates of nonsynonymous substitution in anthropoid primates. Cytochrome b, the mtDNA encoded subunit of ubiquinol-cytochrome c reductase (complex III), also showed an accelerated nonsynonymous substitution rate in anthropoid primates but rate information about the nuclear encoded subunits of complex III has been lacking.We now report that phylogenetic and relative rates analysis of a nuclear encoded catalytically active subunit of complex III, the ironsulfur protein (ISP), shows an accelerated rate of amino acid replacement similar to cytochrome b. Because both ISP and subunit 9, whose function is not directly related to electron transport, are produced by cleavage into two subunits of the initial translation product of a single gene, it is probable that these two subunits of complex III have essentially identical underlying rates of mutation. Nevertheless, we find that the catalytically active ISP has an accelerated rate of amino acid replacement in anthropoid primates whereas the catalytically inactive subunit 9 does not.     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.     

Regulation of respiration and ATP synthesis in higher organisms: Hypothesis

The present view on the regulation of respiration and ATP synthesis in higher organisms implies only Michaelis-Menten type kinetics and respiratory control as regulatory principles. Recent experimental observations, suggesting further regulatory mechanisms at respiratory chain complexes, are reviewed. A new hypothesis is presented implying regulation of respiration and ATP synthesis in higher organisms mainly via allosteric modification of respiratory chain complexes, in particular of cytochromec oxidase. The allosteric effectors, e.g., metabolites, cofactors, ions, hormones, and the membrane potential are suggested to change the activity and the coupling degree of cytochromec oxidase by binding to specific sites at nuclear coded subunits. Recent results on the structure and activity of cytochromec oxidase, supporting the hypothesis, are reviewed.Dedicated to Professor Dr. Carl Martius on the occasion of his 80th birthday.     

A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency

Ubiquinol cytochrome c reductase (complex III) deficiency represents a clinically heterogeneous group of mitochondrial respiratory chain disorders that can theoretically be subject to either a nuclear or a mitochondrial mode of inheritance. In an attempt to elucidate the molecular bases of the disease, we first determined the nucleotide sequence of three unknown subunits (9.5 kDa, 7.2 kDa, 6.4 kDa) by cyberscreening of human expressed sequence tag data bases and sequenced the 11 cDNA subunits encoding complex III in five patients with isolated complex III deficiency. No mutation in the nuclearly encoded complex III subunits was observed, but a mutation in the cd2 helix of the mitochondrial (mt) cytochrome b gene was found to alter the conformation of the bc ₁ complex in one patient with severe hypertrophic cardiomyopathy. The present study is highly relevant to genetic counseling as the absence of mtDNA mutations in all but one patient in our series strongly supports autosomal rather than maternal inheritance in the majority of patients with complex III deficiency. Received: 15 January 1999 / Accepted: 31 March 1999     

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^– parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Defects in mitochondrial respiratory complexes III and IV,and human pathologies

Here, relationships between alterations in tissue-specific content, protein structure, activity, and/or assembly of respiratory complexes III and IV induced by mutations in corresponding genes and various human pathologies are reviewed. Cytochrome bc(1) complex and cytochrome c oxidase (COX) deficiencies have been detected in a heterogeneous group of neuromuscular and non-neuromuscular diseases in childhood and adulthood, presenting a number of clinical phenotypes of variable severity. Such disorders can be caused by mutations located either in mitochondrial genes or in nuclear genes encoding structural subunits of the complexes or corresponding assembly factors/chaperones. Of the defects in mitochondrial DNA genes, mutations in cytochrome b subunit of complex III, and in structural subunits I-III of COX have been described to date. As to defects in nuclear DNA genes, mutations in genes encoding the complexes assembly factors such as the BCS1L protein for complex III; and SURF-1, SCO1, SCO2, and COX10 for complex IV have been identified so far.     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

Defects in the cytochromebc 1 complex in mitochondrial diseases

The clinical and biochemical findings of 14 patients with an isolated defect of thebc ₁ complex have been summarized. The heterogeneity of this group of disorders reflects the severity and tissue specific expression of the defect and the complexity of this multisubunit protein with components that are coded on both nuclear and mitochondrial DNA. The data on several patients with a combined defect of cytochrome oxidase and thebc ₁ complex or with multiple respiratory chain defects have also been presented and discussed in relation to our knowledge of the biosynthesis and assembly of the respiratory chain complexes. The severity of the defectin vivo is illustrated in one patient with isolated complex III deficiency by measurement of O₂ consumption and CO₂ production following exercise, or by³¹P-NMR. The latter also provides a means by which response to therapy can be followed.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.