Evidence for the sequential assembly of cytochrome oxidase subunits in rat liver mitochondria.

The assembly of cytochrome oxidase was studied in isolated rat liver mitochondria and isolated rat hepatocytes labelled in vitro with L-[35S]methionine. This was achieved by studying the temporal association of radioactive subunits which are immunoabsorbed with antibodies against subunits I, II and the holoenzyme. Antibodies against the holoenzyme were shown to be highly specific for subunit V. The results show that subunit I appears in the holoenzyme late in the assembly process. No radioactive subunit I is absorbed with antiserum against subunit II or the holoenzyme (subunit V) after a 30 min pulse in either isolated mitochondria or hepatocytes. However, both antisera absorb radioactive subunits I after a 150 min chase in isolated hepatocytes. This was confirmed using antibodies against subunit I, which absorbed only radioactive subunit I after a 30 min pulse but absorbed radioactive subunits I-III and VI after a 150 min chase. Thus, the late assembly of radioactive subunit I is explained by a temporal sequence in the assembly process and not by the presence of a large, non-radioactive pool of subunit I. Using the above approach and the three specific antisera, the following temporal sequence in the assembly of cytochrome oxidase was established. Subunits II and III assemble rapidly with each other or with cytoplasmically translated subunit VI. This complex of three peptides in turn assembles slowly with subunit I or with the other cytoplasmically translated subunits. The early association of subunit VI with the mitochondrially translated subunits II and III suggests a possible role of the former in integration of the holoenzyme.     

Synthesis of cytochrome oxidase in isolated rat hepatocytes

1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharose-protein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [35S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase.     

Interaction of prostaglandin E1 with human high density lipoproteins

The assembly of cytochrome c oxidase subunits I-III was studied in vitro in isolated rat liver mitochondria pre-labeled with [35S]methionine. Individual subunits were immunoabsorbed with monospecific antibodies. Isolated heme a from rat liver mitochondria, when added to radiolabeled mitochondria, induced assembly of subunit I with subunits II and III. Assembly of these subunits was not observed in mitochondria incubated in the presence of heme b(hemin) or in the absence of heme. Quantitative analysis of immunoabsorbed, radiolabeled subunits suggests that the predominant effect of heme a is on the assembly of subunit I with subunit III.     

Tissue-specific and species-specific distribution of -SH groups in cytochrome c oxidase subunits

Cytochrome c oxidase was isolated from pig, bovine, rat and human tissues including liver, heart, diaphragm and kidney. The native and the sodium-dodecyl-sulfate (SDS)-dissociated enzymes were labelled under optimal conditions with N-ethyl-[2,3-14C]maleimide before and after reduction with dithiothreitol, separated into 13 subunits by SDS gel electrophoresis and the radioactive bands were visualized by fluorography. In some cases the radioactive bands were cut out and counted. All isozymes were labelled in subunits I, III, Va and VIIb, and in subunit II after reduction. Labelling of subunit Vb was equivocal, and in no case were subunits IV and VIc labelled. All other subunits were labelled tissue-specifically and/or species-specifically. No differences were found between labelling of the native and SDS-dissociated enzyme. By relating the molar amount of bound N-ethylmaleimide to the known amount of cysteines in subunits of bovine heart cytochrome c oxidase, the percentage of -SH group reactivity was calculated. Only the cysteine of subunit Va was found to be 100% reactive. The distinct and different reactivity of subunit VIIb as compared to subunits VIIa and VIIc clearly establishes this polypeptide as an independent subunit of mammalian cytochrome c oxidase.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

Hydrophobic photolabelling of the yeast cytochrome c oxidase subunits in contact with lipids

The hydrophobic domain of the membrane-bound enzyme yeast cytochrome c oxidase was labelled with photoactivable phosphatidylcholines.Subunits I, II and III were labelled; a minor labelling was also found on subunits V and VII.The labelling of subunit V was located in a small terminal polypeptide sequence.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. Mitochondrial protein synthesis and analysis of the polypeptide composition of cytochrome c oxidase.

Three previously isolated mutants of Neurospora crassa, temperature-sensitive for the production of cytochrome aa3, have been further analyzed. These mutants have a slightly reduced capacity for mitochondrial protein synthesis when grown at 41 degrees C, although this relative deficiency appeared to be no greater than the deficiency in other cytochrome-aa3-deficient mutants. Thermolability studies revealed that the cytochrome c oxidase purified from each of the mutants grown at 23 degrees C is no more sensitive to heat inactivation than the enzyme isolated from wild-type cells. Sodium dodecylsulfate gel electrophoresis of immunoprecipitates obtained from the mitochondria of each of the mutants grown at 23 degrees C, using antiserum directed against holocytochrome c oxidase, indicated that all the subunits of cytochrome c oxidase were present in relative amounts similar to those found in mitochondria from wild-type cultures. However, when the mitochondria from mutant cultures grown at 41 degrees C were examined in the above fashion, only subunits 5 and 6 of the oxidase were detected. Nonetheless, the mitochondrially synthesized subunit 1, 2 and 3 polypeptides could be immunoprecipitated from mitochondria isolated from mutant cells grown at 41 degrees C and labelled with [3H]leucine in medium containing cycloheximide. Although subunits 4 and 7 could not be detected, because a suitable antibody was not available, the fact that five of the seven subunits were present, but not associated with each other, suggested that the genetic defects in these mutants may affect the process of cytochrome c oxidase assembly.     

Identification of the structural subunits required for formation of the metal centers in subunit I of cytochrome c oxidase of Rhodobacter sphaeroides

Genetic manipulation of the aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides was used to determine the minimal structural subunit associations required for the assembly of the heme A and copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the gene for subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I(a)()) that contained a single heme A. No copper was present in this protein, indicating that the heme a(3)-Cu(B) active site was not assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase forms were synthesized that were copurified by histidine affinity chromatography and separated by anion-exchange chromatography. One form was a highly active subunit I-II oxidase containing a full complement of structurally normal metal centers. This shows that association of subunit II with subunit I is required for stable formation of the active site in subunit I. In contrast, subunit III is not required for the formation of any of the metal centers or for the production of an oxidase with wild-type activity. The second product of the cells lacking subunit III was a large amount of a free form of subunit I that appeared identical to subunit I(a)(). Since significant amounts of subunit I(a)() were also isolated from wild-type cells, it is likely that subunit I(a)() will be present in any preparation of the aa(3)-type oxidase isolated via an affinity tag on subunit I.     

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.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Characterization of two high molecular weight proteins immunoprecipitated with an antibody against rat liver cytochrome c oxidase

Isolated rat hepatocytes were labelled with [35S]methionine, dissolved in Triton X-100-containing buffer, and incubated with antibodies against rat liver cytochrome c oxidase. After separation by dodecyl sulfate-gel electrophoresis the fluorogram of immunoprecipitated proteins showed two labelled bands with apparent molecular weights of 52000 and 182000. The immunological relationship of the two proteins to cytochrome c oxidase was demonstrated by immunocompetition with the isolated enzyme and with purified subunits IV-VIII. Although the precursor nature of the two described proteins for cytoplasmically synthesized subunits of cytochrome c oxidase cannot be excluded, the following observations do not support this assumption: 1) The amount of incorporated radioactivity is too high; 2) they are exclusively located with the microsomal fraction; 3) the turnover is rather slow, compared to that of known precursor proteins.     

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.     

The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase.

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS-polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochrondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.     

Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity

We have investigated the covalent binding of dicyclohexylcarbodiimide (DCCD) to cytochrome c oxidase in relation to its inhibition of ferrocytochrome c-induced H+ translocation by the enzyme reconstituted in lipid vesicles. DCCD bound to the reconstituted oxidase in a time- and concentration-dependent manner which appeared to correlate with its inhibition of H+ translocation. In both reconstituted vesicles and intact beef heart mitochondria, the DCCD-binding site was located in subunit III of the oxidase. The apolar nature of DCCD and relatively minor effects of the hydrophilic carbodiimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, on H+ translocation by the oxidase indicate that the site of action of DCCD is hydrophobic. DCCD also bound to isolated cytochrome c oxidase, though in this case subunits III and IV were labeled. The maximal overall stoichiometries of DCCD molecules bound per cytochrome c oxidase molecule were 1 and 1.6 for the reconstituted and isolated enzymes, respectively. These findings point to subunit III of cytochrome c oxidase having an important role in H+ translocation by the enzyme and indicate that DCCD may prove a useful tool in elucidating the mechanism of H+ pumping.     

Phosphorylation and kinetics of mammalian cytochrome c oxidase

The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biogenesis of the cytochrome bc1 complex in isolated rat hepatocytes

Isolated rat hepatocytes were labeled with [³⁵S]methionine in the absence or presence of cycloheximide or chloramphenicol. The cytochrome $\text{bc}$₁ complex was isolated from labeled cells by a micromethod and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. All subunits except the two smallest, subunits VII and VIII, were labeled in the absence of translational inhibitors. In the presence of cycloheximide only subunit III (molecular weight, 30 000) was labeled. This polypeptide, identified as an apo-cytochrome b, was weakly labeled with [³⁵S]methionine in the presence of cycloheximide, indicating a strict dependence of cytoplasmically synthesized products for its assembly. In the presence of chloramphenicol, labeling was inhibited only in subunit III.