Rapid evolution of cytochrome c oxidase subunit II in camelids (Tylopoda, Camelidae)

Within cetartiodactyl species, both New and Old World camelids are uniquely adapted to the extremely hot and dry climates of African-Asian territories and to the high altitude cold and hypoxic environment of the whole Andean area. In order to investigate the potential association between these particular adaptations and mitochondrial aerobic energy production, we examined the camelid genes of cytochrome c oxidase subunits I, II, and III and the replacement of amino acids inferred. We found that all subunits had undergone a number of replacements in sites otherwise conserved in other cetartiodactyls. Changes of COXI and COXIII were mainly located in the transmembrane helices of proteins. For COXII, although most of the changes did not occur in sites directly involved in electron transfer, a shift of D by T at 115 position of Old World camelid might modify electrostatic interactions with cytochrome c. COXII also showed an increased relative evolutionary rate respect to other cetartiodactyls compared.     

Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer

Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.     

Alternative editing of cytochrome c oxidase III mRNA in trypanosome mitochondria generates protein diversity

Trypanosomes use RNA editing to produce most functional mitochondrial messenger RNA. Precise insertion and deletion of hundreds of uridines is necessary to make full-length cytochrome c oxidase III (COXIII) mRNA. We show that COXIII mRNA can be alternatively edited by a mechanism using an alternative guide RNA to make a stable mRNA. This alternatively edited mRNA is translated to produce a unique protein that fractionates with mitochondrial membranes and colocalizes with mitochondrial proteins in situ. Alternative RNA editing represents a previously unknown mechanism generating protein diversity and, as such, represents an important function for RNA editing.     

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase in inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II-IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site of subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.     

Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation

Mitochondria maintain genome and translation machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. To build up functional enzymes, these organellar gene products must assemble with imported subunits that are encoded in the nucleus. New findings on the early steps of cytochrome c oxidase assembly reveal how the mitochondrial translation of its core component, cytochrome c oxidase subunit 1 (Cox1), is directly coupled to the assembly of this respiratory complex.     

Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues.

Cytochrome c oxidase (EC 1.9.3.1) is an enzyme which is composed of subunits derived from both the mitochondrial and the nuclear genomes. To determine whether or not the expression of these two genomes is co-ordinated at the mRNA level, we have examined the steady-state levels of mRNAs coding for cytochrome c oxidase subunit III (mitochondrially encoded) and subunit VIc (nuclear-encoded) in rat tissues. This was compared with the tissue concentration of the holoenzyme, which was estimated by measuring cytochrome c oxidase enzyme activity. The tissues (heart, brain, liver, kidney, soleus muscle and superficial white vastus muscle) possessed a 13-fold range of enzyme activity, which was highest in heart and lowest in the superficial vastus muscle. Specific subunit mRNA levels were quantified by using slot-blot hybridization of cDNA probes to total tissue RNA. The highest values for subunit III and Vlc mRNA tissue contents were found in kidney, followed by liver and heart (40-60% of that of kidney). The white vastus muscle contained the lowest subunit mRNA level (15% of that of kidney). Although some variability was apparent within each tissue, a parallel pattern of mRNA expression of the nuclear- and mitochondrially encoded subunits was observed. Differences between muscle (heart, vastus and soleus) and non-muscle tissues were noted in the relationship between mRNA and protein levels of expression. Thus, although this suggests that tissue-specific regulatory processes operate, the steady-state expression of subunit III and subunit Vlc mRNAs appears to be co-ordinately regulated.     

The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria.

The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.     

Mechanism of increase in cytochrome c oxidase activity in sweet potato root tissue during aging of slices

The mechanism of an increase in cytochrome c oxidase [EC 1.9.3.1] activity during aging of sliced sweet potato root tissue was investigated with antibiotics and antibody to the purified enzyme. 1. The increase in cytochrome c oxidase activity was inhibited by chloramphenicol but not by cycloheximide. 2. Cytochrome c oxidase purified from wounded tissue was identical with that from intact tissue as judged by the subunit composition, sedimentation velocity, absorption spectrum, antigenicity, and activity per heme a. 3. An increase in the amount of cytochrome c oxidase protein took place during aging of slices. 4. Sweet potato cytochrome c oxidase consists of five subunits. When slices were aged in the presence of [3H]leucine, the three larger subunits (I, II, and III) of cytochrome c oxidase were labeled, while no radioactivity was incorporated into the other two subunits, IV and V. The results indicate that the increase in cytochrome c oxidase activity is due to an increase in the amount of the enzyme protein. We propose that excess amounts of subunits derived from the cytoplasm of the enzyme are present in intact tissue and are assembled with subunits of mitochondrial origin to form the holoenzyme after wounding of tissue.     

Species-specific expression of cytochrome c oxidase subunit III mRNA

1. A cDNA probe encoding cytochrome c oxidase subunit III cloned from rat liver mitochondria was used to quantify mRNA levels in rat, mouse and rabbit tissues. This was compared to its phenotypic expression, using enzyme activity. 2. Enzyme activities were highest in mouse, intermediate in rat, and lowest in rabbit tissues. 3. Subunit III mRNA levels were easily quantified in rat, but could not be accurately measured in rabbit or mouse tissues despite high cytochrome c oxidase activities. 4. Significant subunit III sequence divergence must exist, among these species. Caution should be exercised in quantifying the expression of this mitochondrial gene.     

Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase.

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.     

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.     

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Kinetics of the interaction of the cytochrome c oxidase of Paracoccus denitrificans with its own and bovine cytochrome c

We have devised a relatively simple method for the purification of cytochrome aa3 of Paracoccus denitrificans with three major subunits similar to those of the larger subunits of the mitochondrial cytochrome oxidase. This preparation has no c-type cytochrome. Studies were made of the oxidation of soluble cytochromes c from bovine heart and Paracoccus. The cytochrome-c oxidase activity was stimulated by low concentrations of either cytochrome c, providing an explanation for the multiphasic nature of plots of v/S versus v. Kinetics of the oxidation of bovine cytochrome c by the Paracoccus oxidase resembled those of bovine oxidase with bovine cytochrome c in every way; the Paracoccus oxidase with bovine cytochrome c can serve as an appropriate model for the mitochondrial system. The kinetics of the oxidation of the soluble Paracoccus cytochrome c by the Paracoccus oxidase were different from those seen with bovine cytochrome c, but resembled the latter if poly(L-lysine) was added to the assays. The important difference between the two species of cytochrome c is the more highly negative hemisphere on the side of the molecule way from the heme crevice in the Paracoccus cytochrome. Thus, the data emphasize the importance of all of the charged groups on cytochrome c in influencing the binding or electron transfer reactions of this oxidation-reduction system. The data also permit some interesting connotations about the possible evolution from the bacterial to the mitochondrial electron transport system.     

Cytonuclear coadaptation in Drosophila: disruption of cytochrome c oxidase activity in backcross genotypes

Abstract The cytochrome c oxidase enzyme (COX) is comprised of 10 nuclear-encoded subunits and three mito-chondrial-encoded subunits in close physical association in the inner mitochondrial membrane. COX passes electrons from cytochrome c to molecular oxygen and pumps protons into the inner mitochondrial space for ATP production. Selection on nuclear-mitochondrial interactions within species should lead to coadaptation of the proteins comprising this important enzyme. Under this model, there should be relatively little disruption of COX activity when mitochondrial genomes are crossed among strains within species. A more pronounced disruption of activity is expected when the mitochondrial genome is expressed in the nuclear background of a different species. We test these hypotheses in Drosophila using hybridization and backcrossing among lines of D. simulans and D. mauritiana. Disrupted cytonuclear genotypes were constructed using backcrosses between two lines of D. simulans (siI and si II ) that introduced each divergent mitochondrial DNA (mtDNA) into each nuclear background due to maternal inheritance of mtDNA. Similar crosses were used to introduce eachD. simulans mtDNA into the D. mauritiana maI nuclear background. Reconstituted cytonuclear control genotypes were constructed by backcrossing the initial F1 females to males of the maternal genotype. COX enzyme activities were compared among these disrupted and reconstituted backcross genotypes within and between species. The disruption effect on COX activity was restricted to males of interspecific genotypes. These data support the coadaptation hypothesis and are consistent with predictions that the evolution of modifiers of male mitochondrial dysfunction is hindered by the maternal inheritance of mtDNA. New sequence data for nuclear encoded subunits of COX identified amino acids that may play a role in the disruption effect.     

Human cytochrome c oxidase isoenzymes from heart and skeletal muscle; purification and properties

Human cytochrome c oxidase was isolated in an active form from heart and from skeletal muscle by a fast, small-scale isolation method. The procedure involves differential solubilisation of the oxidase from mitochondrial fragments by laurylmaltoside and KCl, followed by size-exclusion high-performance liquid chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed differences between the subunit VI region of cytochrome c oxidases from human heart and skeletal muscle, suggesting different isoenzyme forms in the two organs. This finding might be of importance in explaining mitochondrial myopathy which shows a deficiency of cytochrome c oxidase in skeletal muscle only. In SDS polyacrylamide gel electrophoresis most human cytochrome c oxidase subunits migrated differently from their bovine counterparts. However, the position of subunits III and IV was the same in the human and in the bovine enzymes. The much higher mobility of human cytochrome c oxidase subunit II is explained by a greater hydrophobicity of this polypeptide than of that of the subunit II of the bovine enzyme.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.