Subunits VIa Research Articles

Destabilization of the Quaternary Structure of Bovine Heart Cytochrome c Oxidase upon Removal of Tightly Bound Cardiolipin

The quaternary structural stability of cardiolipin-containing (CcO(CL+)) versus CL-free cytochrome c oxidase (CcO(CL-)) was compared using structural perturbants as probes. Exposure to increasing concentrations of urea or guanidinium chloride causes sequential dissociation of five subunits from CcO(CL+) in the order VIa and VIb, followed by III and VIIa, and ultimately Vb. Removal of CL from CcO destabilizes the association of each of these five subunits with the core of CcO. Subunits VIa and VIb spontaneously dissociate from CcO(CL-) even in the absence of denaturant and are no longer present after purification of the CL-free 11-subunit complex by ion exchange chromatography. The other 11 subunits remain associated in a partially active complex, but the association of subunits III, VIIa, and Vb is weakened; i.e., the midpoints for the subunit dissociation curves are each shifted to a lower perturbant concentration (lower by 1.1-1.7 M urea; lower by 0.3-0.4 M GdmCl). This corresponds to a decrease of ∼9 kJ in the Gibbs free association energy for each of these subunits when CL is removed from CcO. With either CcO(CL+) or CcO(CL-), loss of enzymatic activity occurs coincident with dissociation of subunits III and VIIa. The loss of activity is irreversible, and reactivation of CcO(CL-) by exogenous CL occurs only if both subunits remain associated with the core of CcO. Inclusion of sulfate anions stabilizes the association of VIIa more than III, resulting in a slight separation of the urea-induced dissociation curves. In this case, activity loss correlates much better with dissociation of subunit VIIa than III. We conclude that (1) bound cardiolipin is an important stabilizing factor in the quaternary structure of CcO and (2) association of subunit VIIa (possibly together with subunit III) is critical for functional CL binding and full electron-transfer activity of CcO.

Ferricytochrome c protects mitochondrial cytochrome c oxidase against hydrogen peroxide-induced oxidative damage

An excess of ferricytochrome c protects purified mitochondrial cytochrome c oxidase and bound cardiolipin from hydrogen peroxide-induced oxidative modification. All of the peroxide-induced changes within cytochrome c oxidase, such as oxidation of Trp 19,IV and Trp 48,VIIc, partial dissociation of subunits VIa and VIIa, and generation of cardiolipin hydroperoxide, no longer take place in the presence of ferricytochrome c. Furthermore, ferricytochrome c suppresses the yield of H 2O 2-induced free radical detectable by electron paramagnetic resonance spectroscopy within cytochrome c oxidase. These protective effects are based on two mechanisms. The first involves the peroxidase/catalase-like activity of ferricytochrome c, which results in the decomposition of H 2O 2, with the apparent bimolecular rate constant of 5.1 ± 1.0 M − 1 s − 1 . Although this value is lower than the rate constant of a specialized peroxidase, the activity is sufficient to eliminate H 2O 2-induced damage to cytochrome c oxidase in the presence of an excess of ferricytochrome c. The second mechanism involves ferricytochrome c-induced quenching of free radicals generated within cytochrome c oxidase. These results suggest that ferricytochrome c may have an important role in protection of cytochrome c oxidase and consequently the mitochondrion against oxidative damage.

Nuclear Respiratory Factor 1 Controls Myocyte Enhancer Factor 2A Transcription to Provide a Mechanism for Coordinate Expression of Respiratory Chain Subunits

Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. We used genome comparisons to conclude that the promoter regions of COX6A(H) and COX7A(H) lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. We show that MEF2A mRNA is induced with forced expression of NRF1 and that the MEF2A 5'-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, we used an isoform-specific antibody in ChIP to confirm MEF2A binding to the COX6A(H) promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 --> MEF2A --> COX(H) transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1alpha in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. Our findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4.

Differential Stability of Dimeric and Monomeric Cytochrome c Oxidase Exposed to Elevated Hydrostatic Pressure

Detergent-solubilized dimeric and monomeric cytochrome c oxidase (CcO) have significantly different quaternary stability when exposed to 2-3 kbar of hydrostatic pressure. Dimeric, dodecyl maltoside-solubilized cytochrome c oxidase is very resistant to elevated hydrostatic pressure with almost no perturbation of its quaternary structure or functional activity after release of pressure. In contrast to the stability of dimeric CcO, 3 kbar of hydrostatic pressure triggers multiple structural and functional alterations within monomeric cytochrome c oxidase. The perturbations are either irreversible or slowly reversible since they persist after the release of high pressure. Therefore, standard biochemical analytical procedures could be used to quantify the pressure-induced changes after the release of hydrostatic pressure. The electron transport activity of monomeric cytochrome c oxidase decreases by as much as 60% after exposure to 3 kbar of hydrostatic pressure. The irreversible loss of activity occurs in a time- and pressure-dependent manner. Coincident with the activity loss is a sequential dissociation of four subunits as detected by sedimentation velocity, high-performance ion-exchange chromatography, and reversed-phase and SDS-PAGE subunit analysis. Subunits VIa and VIb are the first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the resulting 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO containing all 13 subunits. However, dimeric CcO, in which the association of VIa and VIb is stabilized, is not susceptible to pressure-induced inactivation. We conclude that dissociation of subunit III and/or VIIa must be responsible for pressure-induced inactivation of CcO since VIa and VIb can be removed from monomeric CcO without significant activity loss. These results are the first to clearly demonstrate an important structural role for the dimeric form of cytochrome c oxidase, i.e., stabilization of its quaternary structure.

Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase

The lipid-soluble peroxides, tert-butyl hydroperoxide and peroxidized cardiolipin, each react with bovine cytochrome c oxidase and cause a loss of electron-transport activity. Coinciding with loss of activity is oxidation of Trp 19 and Trp 48 within subunits VIIc and IV, and partial dissociation of subunits VIa and VIIa. tert-Butyl hydroperoxide initiates these structural and functional changes of cytochrome c oxidase by three mechanisms: (1) radical generation at the binuclear center; (2) direct oxidation of Trp 19 and Trp 48; and (3) peroxidation of bound cardiolipin. All three mechanisms contribute to inactivation since blocking a single mechanism only partially prevents oxidative damage. The first mechanism is similar to that described for hydrogen peroxide [ Biochemistry 43:1003–1009; 2004], while the second and third mechanism are unique to organic hydroperoxides . Peroxidized cardiolipin inactivates cytochrome c oxidase in the absence of tert-butyl hydroperoxide and oxidizes the same tryptophans within the nuclear-encoded subunits. Peroxidized cardiolipin also inactivates cardiolipin-free cytochrome c oxidase rather than restoring full activity. Cardiolipin-free cytochrome c oxidase, although it does not contain cardiolipin, is still inactivated by tert-butyl hydroperoxide, indicating that the other oxidation products contribute to the inactivation of cytochrome c oxidase. We conclude that both peroxidized cardiolipin and tert-butyl hydroperoxide react with and triggers a cascade of structural alterations within cytochrome c oxidase. The summation of these events leads to cytochrome c oxidase inactivation.

Protein Kinase A-mediated Phosphorylation Modulates Cytochrome c Oxidase Function and Augments Hypoxia and Myocardial Ischemia-related Injury

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Photolabeling of Cardiolipin Binding Subunits within Bovine Heart CytochromecOxidase

Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues were synthesized: (1) 2-SAND-gly-CL with a nitrophenylazido group attached to the polar headgroup of cardiolipin (CL) via a linker containing a cleavable disulfide; (2) 2',2''-bis-(AzC12)-CL with two of the four fatty acid tails of cardiolipin replaced by 12-(N-4-azido-2-nitrophenyl) aminododecanoic acid. Both arylazido-CL derivatives were used to map the cardiolipin binding sites within two types of detergent-solubilized CcO: (1) intact 13-subunit CL-containing CcO (three to four molecules of endogenous CL remain bound per CcO monomer); (2) 11-subunit CL-free CcO (subunits VIa and VIb are missing because they dissociate during CL removal). Upon the basis of these photolabeling studies, we conclude that (1) subunits VIIa, VIIc, and possibly VIII are located near the two high-affinity cardiolipin binding sites, which are present in either form of CcO, and (2) subunit VIa is located adjacent to the lower affinity cardiolipin binding site, which is only present in the 13-subunit form of CcO. These data are consistent with the recent CcO crystal structure in which one cardiolipin is located near subunit VIIa and a second is located near subunit VIa (PDB ID code referenced in Tomitake, T. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15304-15309). However, we propose that a third cardiolipin is bound between subunits VIIa and VIIc near the entrance to the D-channel. Cardiolipin bound at this location could potentially function as a proton antenna to facilitate proton entry into the D-channel. If true, it would explain the CcO requirement of bound cardiolipin for full electron transport activity.

Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide.

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.

Cholate-induced dimerization of detergent- or phospholipid-solubilized bovine cytochrome C oxidase.

Bovine heart cytochrome c oxidase (CcO), solubilized by either nonionic detergents or phospholipids, completely dimerizes upon the addition of bile salts, e.g., sodium cholate, sodium deoxycholate, or CHAPS. Bile salt induced dimerization occurs whether dodecyl maltoside, decyl maltoside, or Triton X-100 is the primary solubilizing detergent or the enzyme is dispersed in phosphatidylcholine, phosphatidylethanolamine, or mixtures thereof. In each case, complete CcO dimerization can be verified by sedimentation velocity and sedimentation equilibrium after correction for bound detergent and/or phospholipid. The relative concentration of the bile salt is critical for production of homogeneous, dimeric CcO. For example, enzyme solubilized by 2 mM detergent requires an equal molar concentration of sodium cholate. Similarly, enzyme dispersed in 20 mM phospholipid requires 50 mM sodium cholate, concentrations that are commonly used to reconstitute CcO into small unilamellar vesicles. Bile salts do more than just stabilize dimeric CcO and prevent detergent-induced dissociation into monomers. They are able to completely reverse detergent-induced monomerization and cause completely monomeric CcO to reassociate. Dimeric CcO so generated is no more stable than the original complex and easily dissociates into monomers if the bile salt is removed. The dimerization process is dependent upon a full complement of subunits; e.g., if subunits VIa and VIb are removed, the resulting monomeric CcO will not reassociate upon the addition of sodium cholate. These results support four important consequences: (1) dissociation of dimeric CcO into monomers is reversible; (2) stable dimers can be produced under solution conditions; (3) dimers can be stabilized even at relatively high pH and low enzyme concentration; and (4) subunits VIa and VIb are required for dimerization.

Characterization of the murine gene for subunit VIIaL of cytochrome c oxidase

Mammalian cytochrome c oxidase consists of thirteen subunits, ten encoded by the nuclear genome and three by the mitochondrial DNA. In several species, two isoforms have been isolated for nuclear-encoded subunits VIa, VIIa and VIII : an ubiquitous L (liver) form and a heart- and skeletal-muscle specific H form. The gene for murine cytochrome c oxidase subunit VIIa-L ( Cox7aL) and its promoter region were isolated, sequenced and analysed. The coding region is split in four exons spanning 4.1 kbp and the promoter carries potential binding sites for Sp1, NRF1 and NRF2 transcription factors. Transcriptional activity of the promoter in reporter assays suggested an ubiquitous expression in mouse tissues.

Phospholipase A(2) digestion of cardiolipin bound to bovine cytochrome c oxidase alters both activity and quaternary structure.

Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.

A Missense Mutation of Cytochrome Oxidase Subunit II Causes Defective Assembly and Myopathy

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

Assembly of cytochrome-c oxidase in cultured human cells.

The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.

Influence of thyroid hormone on the tissue-specific expression of cytochrome c oxidase isoforms during cardiac development

In mammals, cytochrome c oxidase (COX) is composed of 13 different protein subunits. In the rat, two nuclear-encoded subunits, COX VIa and VIII, exist as tissue-specific isoforms: heart and liver. Using Northern-blot analysis, the levels of transcripts for the heart and liver isoforms of VIa and VIII were examined in developing rat hearts. The liver isoform was found to be the predominant form of subunit VIa and the exclusive form of VIII in the 18-day fetal hearts. The mRNA levels of the heart isoform of both subunits increased dramatically to reach adult levels by 14 days. Although the levels of the VIa- and VIII-liver isoform mRNAs remained stable throughout early development, their levels decreased by 40 and 36% respectively between the 18-day fetal stage and 18-day neonatal stage. Therefore the up-regulation of the heart isoforms and down-regulation of the liver isoforms appear to be regulated in a co-ordinated manner during development. To determine if thyroid hormone influences the expression of these developmentally regulated isoforms, the RNA was also extracted from the hearts of 2-week-old hypothyroid rats. The results showed that the levels of VIII-heart and VIa-liver COX mRNAs were approx. 40% lower in the hypothyroid hearts, while VIII-liver and VIa-heart COX isoform expression remained unchanged. These data demonstrate that the isoforms of COX subunits VIa and VIII are not co-ordinately regulated by changes in thyroid hormone levels. Therefore we conclude that, although thyroid hormone influences the expression of isoforms, it appears to do so via a different mechanism from that which regulates the developmental transition.

Separation and Quantitation of CytochromecOxidase Subunits by Mono-Q Fast Protein Liquid Chromatography and C18 Reverse-Phase High-Performance Liquid Chromatography

Mono-Q fast protein liquid chromatography (FPLC) combined with C18 reverse-phase HPLC was used for quantitative subunit analysis of bovine heart cytochromecoxidase, a multisubunit membrane complex. By this approach normal cytochromecoxidase preparations were shown to be a mixture of enzyme that has all 13 subunits and complexes that are missing 1–3 subunits. A distinct advantage of this procedure is that homogeneous 13- or 11-subunit enzyme can be easily isolated from heterogeneous cytochromecoxidase mixtures. The method involves: (1) separation of complexes that are depleted of subunits using Mono-Q FPLC and (2) quantitative subunit analysis of the purified complexes by C18 reverse-phase HPLC with a water/acetonitrile gradient in 0.1% trifluoroacetic acid. The approach has four distinct advantages over other methods of analysis, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) or C4 reverse-phase HPLC. (1) The reproducible yield and the baseline resolution between each eluting subunit permits quantitative determination of the subunit content with an accuracy of ± 5%. (2) Subunits that are very difficult to separate by SDS–PAGE, e.g., subunits VIa, VIb, and VIc, are completely resolved by this system. (3) The combination of Mono-Q purification and C18 reverse-phase HPLC analysis permits an accurate assessment of both homogeneity and subunit content. (4) The quantitative nature of the reverse-phase HPLC system also makes it a powerful method for analyzing the specificity and extent of chemical modification of specific subunits as is shown by the difference in reac tivity of subunit VIa towardN-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate and 4,4′-dipyridyl disulfide.

Species-specific expression of cytochrome c oxidase isozymes

Cytochrome c oxidase was isolated from livers and hearts of sheep, dog and rabbit, and the polypeptide composition was analyzed by two different SDS-PAGE separation systems. The gels were blotted on PVDF-membranes and the N-terminal amino acid sequences of the tissue-specific subunits VIa, VIIa and VIII were determined in a protein sequencer. Except for subunit VIIa from rat, subunits VIa and VIIa from all investigated mammals are tissue-specific expressed in liver and heart. In contrast, subunit VIII is clearly different in liver and heart of bovine, dog and rat, but identical in liver and heart of human (liver-type), sheep, rabbit and also in rainbow trout (heart-type). The data suggest a strong species-specific variation of the regulatory properties of cytochrome c oxidase in different tissues.

Cytochrome‐c Oxidase in Developing Rat Heart Enzymic Properties and Amino‐terminal Sequences Suggest Identity of the Fetal Heart and the Adult Liver Isoform

Perinatal development of cytochrome-c oxidase (complex IV) and ubiquinol-cytochrome-c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densitometrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223-231]. A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.

Enzymatic activities of mitochondrial respiratory complexes from children muscular biopsies. Age-related evolutions

Measurements were performed to determine maximum enzymatic activities of citrate synthetase and respiratory complexes I, III, and IV of mitochondria obtained from muscular biopsies in control children. The significant number of determinations carried out (43 different biopsies in controls aged 3.8 to 19.1 years) permits the formulation of a table of statistically validated reference values for these activities. These values are independent of sex of the controls, and of the studied muscles. Citrate synthetase activity, which remains stable in this age range, thus constitutes a good internal indicator of mitochondrial activity. Complexes I and III manifest activity which does not vary with age. On the other hand, cytochrome oxidase activity shows a highly significant decrease in this age group. This decrease may be correlated with qualitative changes (subunits VIa and VIIa) in composition of this complex.

Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme.

Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20 degrees C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolated in lauryl maltoside contains these same nine different polypeptides and three others, including homologues of subunits VIa and VIb of the mammalian enzyme.

Steady-state transcript levels of cytochrome c oxidase genes during human myogenesis indicate subunit switching of subunit VIa and co-expression of subunit VIIa isoforms

Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the β subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so-called liver-type form of subunit VIa of cytochrome c oxidase diminished during the course of differentiation, while transcription of the so-called heart-type form was induced. Transcripts for the liver-type form and for the heart-type form of subunit VIIa of cytochrome c oxidase were detected in all myogenic cultures; the levels of the heart-type form progressively increased during the course of differentiation. The levels of the other transcripts studied did not change substantially. The results suggest subunit switching of subunit VIa and co-expression of subunit VIIa isoforms during myogenesis. The differential changes in mRNA levels of the heart-type subunits VIa and VII a and the differential changes in mRNA levels of the liver-type subunits VIa and VIIa demonstrate that different transcriptional regulation mechanisms are present for both heart-type genes as well as for both liver-type genes.