Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria

Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.     

Biophysical and biochemical characterization of reconstituted and purified Rhodobacter sphaeroides cytochrome c oxidase in phospholipid vesicles sheds insight into its functional oligomeric structure

Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme.     

The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis

The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the Cu_A-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of Cu_A-site formation.     

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]     

Modified structure and kinetics of cytochrome-c oxidase in fibroblasts from patients with Leigh syndrome

In this study we compared the properties of cytochrome-c oxidase (COX) in cultured fibroblasts from two patients with Leigh Syndrome with COX from control fibroblasts. The fibroblasts from patients showed decreased growth reates and elevated lactate production. COX activity of patients fibroblasts was about 25% of control. Kinetic studies with isolated mitochondria showed a higher K_m for cytochrome c and a markedly reduced molecular turnover of COX from patients, indicating a different structure of the enzyme. A biphasic change of COX activity was obtained by titration of dodecylmaltoside solubilized mitochondria from control fibroblasts with increasing concentrations of anions. With patient mitochondria we found only the inhibiting phase of COX activity and, in contrast to control mitochondria, irreversible inhibition of COX activity by guanidinium chloride. ELISA titrations with monoclonal antibodies to subunit II, IV, Vab, VIac and VIIab indicated a normal amount of mitochondrial coded subunit II, but a reduced amound of nuclear coded subunits. The data indicate incompletely assembled nuclear coded subunits of COX from patient fibroblasts.     

Biochemical and biophysical properties of purified phospholipid vesicles containing bovine heart cytochrome c oxidase

Liposomes containing bovine heart cytochrome c oxidase (COV) prepared by the cholate dialysis technique were purified from those devoid of the enzyme using discontinuous sucrose density ultra centrifugation to eliminate interference in proton-pumping assays. This technique was also used to purify liposomes containing cytochrome c oxidase depleted in subunit III (COV-III), a COX enzyme preparation with altered subunit structure, to assess if the technique could be applied to COX enzymes in which structural and functional changes have occurred. Upon discontinuous sucrose density ultra gradient ultracentrifugation, either COV or COV-III were separated into two bands. Liposomes devoid of enzyme sedimented into the 12% sucrose layer, whereas enzyme-containing liposomes (pCOV or pCOV-III) were found in the 13% sucrose layer. The yield of both pCOV or pCOV-III was greater than 60% (based on heme aa(3) content), suggesting a similar distribution of cytochrome c oxidase (COX) and subunit III-depleted enzyme (COX-III) in the purified liposomes. The number of COX or COX-III molecules per phospholipid vesicle in purified fractions was estimated to be two. Removal of subunit III (M(r)=29,918) from COX resulted in a 30% decrease in electron transfer activity (either in COV-III or pCOV-III) when compared with COV and pCOV, respectively. Both pCOV and pCOV-III exhibited low endogenous proton permeability, as assessed by possessing high respiratory control ratios (14 and greater) and by having similar valinomycin concentration dependencies for stimulation of electron transfer activity in the presence of saturating amounts of CCCP. COV-III and pCOV-III exhibited a 39-44% decrease in proton-pumping activity when compared with COV and pCOV. These results showed that the separation of COX containing liposomes from those lacking enzyme by sucrose density gradient centrifugation can be used to characterize the biophysical properties of these liposomes.     

Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry

Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O₂-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.     

Tracing hybrid incompatibilities to single amino acid substitutions

Deleterious interactions among genes cause reductions in fitness of interpopulation hybrids (hybrid breakdown). Identifying genes involved in hybrid breakdown has proven difficult, and few studies have addressed the molecular basis of this widespread phenomenon. Because proper function of the mitochondrial electron transport system (ETS) requires a coadapted set of nuclear and mitochondrial gene products, ETS genes present an attractive system for studying the evolution of coadapted gene complexes within isolated populations and the loss of fitness in interpopulation hybrids. Here we show the effects of single amino acid substitutions in cytochrome c (CYC) on its functional interaction with another ETS protein, cytochrome c oxidase (COX) in the intertidal copepod Tigriopus californicus. The individual and pairwise consequences of three naturally occurring amino acid substitutions in CYC are examined by site-directed mutagenesis and found to differentially effect the rates of CYC oxidation by COX variants from different source populations. In one case, we show that interpopulation hybrid breakdown in COX activity can be attributed to a single naturally occurring amino acid substitution in CYC.     

Impact of the invasive plant species “Nicotiana glauca” toxins on the larvae of the invasive insect species “Rhynchophorus ferrugineus”: A damaging pest of date palm trees in Saudi Arabia

The wild tree tobacco (Nicotiana glauca) is an alien species that invaded vast areas of the Southwestern region of Saudi Arabia. While, the Red Palm Weevil (RPW) (Rhynchophorus ferrugineus) is considered to be the most damaging invasive insect species of palm trees all over the kingdom of Saudi Arabia, causing major economic losses to farmers and the economy of the country. Using conventional insecticides to control harmful insects such as RPW has undesirable effects on the environment and human health. Alternatively, using biocontrol agents such as poisonous extracts from N. glauca might be a better approach in pest management and can be considered as an eco-friendly, cost-effective, and safe alternative. Therefore, the current study aimed to evaluate the larvicidal effect of N. glauca aqueous extracts against the red palm weevil larvae. The plant specimens were collected from Al-Baha region in the Southwest of Saudi Arabia. Each single test consisted of 20 larvae, and N. glauca preparations were; 1, 1.5, 2, 2.5, and 3 ml, besides the control test. Results obtained for the effect of botanical extracts; leaf, flower, stem and root against R. ferrugineus larvae for an exposure period of 24 hr. at the concentrations of 2.8, 4.2, 6.0, 7.0 and 8.0 ppm. The concentrations for N. glauca extracts reflected an LC₅₀ of 2.7 ppm for leave, 2.6 ppm for flower, 2.8 ppm for stem and 7.00 ppm for root. While, the same concentrations extracts reflected an LC₉₅of 11 ppm for leaf, 9.6 ppm for flower, 8.9 ppm for stem and 13.00 ppm for root. These results showed that N. glauca extracts have a remarkable potentiality as insecticidal substances that can be used as an ecofriendly integrated approach for the management of R. ferrugineus.     

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.     

The characterization and role of zinc binding in yeast Cox4

Yeast Cox4 is a zinc binding subunit of cytochrome c oxidase. Cox4 is the only cofactor-containing subunit that is not directly part of the catalytic core of the enzyme located in the mitochondrial inner membrane. The Zn(II) site is shown to be distinct from the bovine ortholog, as it results from the x-ray structure of the entire cytochrome c oxidase in having a single histidyl residue and three conserved cysteines residues in the coordination sphere. Substitutions at the Cys ligand positions result in non-functional Cox4 proteins that fail to lead to cytochrome oxidase assembly. Limited function exists in His-119 mutants when overexpressed. Zn(II) binding in Cox4 is, therefore, important for the stability of the complex. The solution structure of yeast Cox4 elucidated by multidimensional NMR reveals a C-terminal globular domain consisting of two beta sheets analogous to the bovine ortholog except the loop containing the coordinating His in the yeast protein and the fourth Cys in the bovine protein are in different positions in the two structures. The conformation of this loop is dictated by the different sequence position of the fourth coordinating zinc ligand. The Zn(II) ion is buried within the domain, consistent with its role in structural stability. Potential functions of this matrix-facing subunit are discussed.     

Association of Cytochrome c with Membrane-Bound Cytochrome c Oxidase Proceeds Parallel to the Membrane Rather Than in Bulk Solution

Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c₅₅₂, and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions.     

Cytochrome oxidase assembly does not require catalytically active cytochrome C

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

Knockdown of Human COX17 Affects Assembly and Supramolecular Organization of Cytochrome c Oxidase

Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel ∼ 150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated synthesis and association of individual complexes, is proposed.     

Cytochrome oxidase assembly in yeast requires the product of COX11, a homolog of the P. denitrificans protein encoded by ORF3.

The synthesis of cytochrome oxidase in Saccharomyces cerevisiae was recently shown to require a protein encoded by the nuclear gene COX10. This protein was found to be homologous to the putative protein product of the open reading frame ORF1 reported in one of the cytochrome oxidase operons of Paracoccus denitrificans. In the present study we demonstrate the existence in yeast of a second nuclear gene, COX11, whose encoded protein is homologous to another open reading frame (ORF3) present in the same operon of P. denitrificans. Mutations in COX11 elicit a deficiency in cytochrome oxidase. In this and in other respects cox11 and cox10 mutants have very similar phenotypes. An antibody has been obtained against the yeast COX11 protein. The antibody recognizes a 28 kd protein in yeast mitochondria, consistent with the size of the protein predicted from the sequence of COX11. The COX11 protein is tightly associated with the mitochondrial membrane but is not a component of purified cytochrome oxidase. An analysis of cytochrome oxidase subunits in wild type and in a cox11 mutant suggests that the COX11 protein is not required either for synthesis or transport of the subunit polypeptides into mitochondria. It seems more probable that COX11 protein exerts its effect at some terminal stage of enzyme synthesis, perhaps in directing assembly of the subunits.     

Liposomal and Mitochondrial Cytochrome Oxidase Display Similar Bioenergetic Properties

Abstract

Cytochrome c oxidase, the terminal electron acceptor of the respiratory chain of mitochondria, is an integral membrane protein. The bioenergetic properties of cytochrome oxidase can be studied only when the macromolecule is inserted in a phospholipid bilayer, either in situ or after reconstitution into liposomal membranes. Reintegration of purified cytochrome oxidase in liposomes allows quantitative tests of mechanistic hypothesis concerning the functional properties of the enzyme. Small unilamellar vesicles are prepared by sonication of purified soybean asolectin, and reconstitution of cytochrome oxidase in the bilayer is carried out according to the cholate/dialysis procedure. The proteoliposomes are shown to mimick the mitochondrial state of the enzyme in so far as liposomal cytochrome oxidase : a) displays the same vectorial orientation, the cytochrome c binding site being externally exposed, b) pumps protons in the physiological inside/outside direction, and c) is functionally controlled by the transmembrane electrochemical gradient, i.e. displays respiratory control.     

COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to pet complementation group G19 lack cytochrome oxidase activity and cytochromes a and a3. The enzyme deficiency is caused by recessive mutations in the nuclear gene COX10. Analyses of cytochrome oxidase subunits suggest that the product of COX10 provides an essential function at a posttranslational stage of enzyme assembly. The wild type COX10 gene has been cloned by transformation of a mutant from complementation group G19 with a yeast genomic library. Based on the nucleotide sequence of COX10, the primary translation product has an Mr of 52,000. The amino-terminal 190 residues constitute a hydrophilic domain while the carboxyl-terminal region is hydrophobic and has nine potential membrane-spanning segments. The sequence of the carboxyl-terminal hydrophobic region is homologous to an unidentified protein encoded by a reading frame (ORF1) located in one of the cytochrome oxidase operons of Paracoccus denitrificans. The two proteins share 24% identical residues and exhibit very similar hydrophobicity profiles. The bacterial homolog, however, lacks the hydrophilic amino-terminal region of the yeast protein.     

Genetic variation and subspecific status of the grey wolf (Canis lupus) in Saudi Arabia

This work represents the most extensive genetic study of the grey wolf (Canis lupus Linnaeus, 1758) in Arabia and the first considering genetic data from multiple locations within Saudi Arabia. Previous suggestion of the occurrence of two subspecies of wolves in Arabia is not supported by this study. The genetic evidence suggests that the wolves of Saudi Arabia are genetically variable and more closely related to the Eurasian wolf Canis lupus group (dog included) than to the Indian wolf C. l. pallipes. The genetic diversity observed for C. lupus in Saudi Arabia indicates that the subspecific status C. l. arabs should be retained for the Arabian wolf. What remains unclear is the degree to which genetic introgression from domestic dogs has influenced the composition and integrity of C. lupus in Saudi Arabia.