Cytochrome c oxidase subunits in contact with phospholipids. Hydrophobic photolabeling with azidophospholipids.

The technique of photolabeling of membrane proteins with arylazidophospholipids was applied to cytochrome c oxidase. The "deep" and "shallow" labels employed reacted with all subunits of cytochrome c oxidase except V and VI: Subunits I, III, and VII were heavily labeled, Subunit II was labeled to a lesser extent, and Subunit IV was poorly labeled. Subunit I was labeled more by the deep label and Subunit VII by the shallow one. The other subunits were equally labeled by the two probes. This technique has revealed what subunits of cytochrome c oxidase interact with the lipid and their approximate position in the membrane.     

Structure of the cytochrome c oxidase complex: labeling by hydrophilic and hydrophobic protein modifying reagents

Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase.     

Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity.

In order to study the role of the individual subunits of yeast cytochrome c oxidase, rabbit antisera were prepared against Subunit II (a mitochondrially made polypeptide) and Subunit VI (a cytoplasmically made polypeptide). Antisera were also obtained against a mixture of the two mitochondrially made subunits (I PLUS II) and against mixtures of the following cytoplasmically made subunits: (IV PLUS VI); (V PLUS VII); and (IV PLUS V PLUS VI PLUS VII). Neither anti-II serum nor anti-VI serum cross-reacted with any of the other six subunits of cytochrome c oxidase as judged by a sensitive ring test or by double diffusion in agarose gels. Anti-II serum inhibited the oxidation of ferrocytochrome c by purified yeast cytochrome c oxidase or by freshly isolated as well as sonically fragmented yeast mitochondria. Anti-(V, VII) serum and anti-(IV, V, VI, VII) serum were also strongly inhibitory. Anti-VI serum and anti-(IV, VI) serum inhibited only weakly. If purified cytochrome c oxidase was inhibited with a saturating amount of anti-VI serum, anti-II serum elicited a further increment of inhibition, as would be expected if the inhibitory effects of these two antisera involved different antigenic sites on the holoenzyme. Each of the antisera precipitated all seven cytochrome c oxidase subunits from crude mitochondrial extracts. However, anti-VI and, particularly, anti-II were much less effective precipitants than antisera against Subunits IV to VII or antisera against the holoenzyme. These data suggest that the oxidation of ferrocytochrome c by cytochrome c oxidase required both mitochondrially as well as cytoplasmically made subunits.     

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.     

Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.     

Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.     

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.     

The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.     

Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits.

Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes. The eukaryotic enzyme complex consists of a large number of polypeptide subunits. Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded. Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane. Subunits I and II are the catalytic core of the enzyme. Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site. Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme. Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes. Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative. We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro. Whether this is a physiological control mechanism remains to be seen.     

Heme is necessary for the accumulation and assembly of cytochrome c oxidase subunits in Saccharomyces cerevisiae.

The presence of cytochrome c oxidase subunits and the association of these subunits with each other was studied in a heme-deficient Saccharomyces cerevisiae mutant. This mutant had been isolated by Gollub et al. (1977) J. Biol. Chem. 252, 2846-2854) and had been shown lack delta-aminolevulinic acid synthetase. When grown in the absence of heme or heme precursors, the mutant is respiration-deficient, devoid of cytochrome absorption bands and auxotrophic for all those components whose biosynthesis is dependent on hemoproteins; when grown in the presence of heme or heme precursors, the mutant is phenotypically wild type. Upon growth of the mutant in the absence of heme synthesis, the mitochondria still contained two of the three mitochondrially made cytochrome c oxidase subunits (i.e. II and III) and at least one of the cytoplasmically made cytochrome c subunits (VI). The other subunits were either barely detectable (I, IV) or undetectable (V, VII). The residual subunits were apparently not assembled with each other since an antiserum directed mainly against Subunit VI failed to co-precipitate Subunits II and III which were still present. In contrast, growth of the mutant in the presence of delta-aminolevulinic acid led to the accumulation of active, fully assembled cytochrome c oxidase in the mitochondria. Heme a (or one of its precursors) thus controls the assembly of cytochrome c oxidase from its individual subunits.     

Cytochrome c oxidase from bakers' yeast. III. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides.

Earlier studies have shown that cytochrome c oxidase from bakers' yeast is an oligomeric enzyme which contains three polypeptides (I to III) synthesized on mitochondrial ribosomes and four polypeptides (IV to VII) synthesized on cytoplasmic ribosomes. These polypeptide subunits have now been isolated by a simple protocol which utilizes differences in polypeptide charge, solubility, and size. Their molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, gel filtration in the presence of guanidine hydrochloride, and amino acid analysis were: I, 40,000; II, 33,000; III, 22,000; IV, 14,500; V, 12,700; VI, 12,700; and VII, 4,600. All seven polypeptide subunits exhibited acidic isoelectric points; cytoplasmically made subunits were more acidic than mitochondrially made ones. The amino acid composition of two mitochondrially made subunits and two cytoplasmically made subunits was determined. The two mitochondrial translation products, I and II, contained only 34.7% and 42.1% polar amino acids, respectively, whereas the two cytoplasmic translation products, IV and VI, contained 48.3% and 49.3%, respectively. This agreed with the observation that Subunits I and II are very insoluble, requiring detergents for solubility, whereas Subunits IV and VI are water-soluble in the absence of any added detergent. These results indicate that the cytochrome c oxidase subunits synthesized on mitochondrial and cytoplasmic ribosomes are fundamentally different in size, isoelectric properties, and hydrophobicity. They also suggest the possibility that at least some of the mitochondrially made subunits are buried in the lipid phase of the mitochondrial inner membrane.     

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.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Cytochrome c oxidase from bakers' yeast. V. Arrangement of the subunits in the isolated and membrane-bound enzyme.

Cytochrome c oxidase from baker's yeast contains three mitochondrially made subunits (I to III) which are relatively hydrophobic and four cytoplasmically made subunits (IV to VII) which are relatively hydrophilic (Mason, T. L., Poyton, R. O., Wharton, D.C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354 and Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761). In order to explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of "surface probes" was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound cytochrome c oxidase. The surface probes included iodination with lactoperoxidase and coupling with p-diazonium benzenesulfonate. In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound isocyanate group. In the membrane-bound enzyme, Subunit I was almost completely inaccessible and Subunit II was partly inaccessible to all surface probes. All of the other subunits were accessible. Similar results were obtained with the solubilized enzyme, except that the differences in reactivity between the individual subunits were less clear-cut. The results obtained with liposome-bound cytochrome c oxidase resembled those obtained with the mitochondrially bound enzyme. These data suggest that the two largest mitochondrially made subunits are localized in the interior of the enzyme and that they are genuine components of cytochrome c oxidase.     

The biogenesis of a cytochrome b complex in yeast mitochondria: Sites of translation of the protein components

A spectrally pure cytochrome b complex has been isolated from yeast mitochondria and shown to contain seven nonidentical subunits with the following molecular weights: I, 42,000; II, 33,000; III, 27,500; IV, 23,000; V, 15,500; VI, 13,000; and VII, 10,500. In order to determine the intracellular sites of translation of these polypeptides, yeast cells were labeled with [³H]leucine in the presence of specific inhibitors of mitochondrial or cytoplasmic translation. The labeling of subunits I and III was found to be insensitive to cycloheximide but was inhibited by chloramphenicol. Alternatively, subunits IV–VII were labeled in the presence of chloramphenicol but not in the presence of cycloheximide. Since subunit II was not significantly labeled in the presence of either inhibitor, the technique of labeling in vivo with [³H]formate was used to establish its site of biogenesis. Formate is incorporated by mitochondrial, but not cytoplasmic, ribosomes as N-formylmethionine at initiation and is therefore a marker for the products of mitochondrial translation. Subunits I–III were labeled under these conditions whereas the four smallest subunits were not. Taken together, the findings clearly establish that the three largest subunits of the cytochrome b complex are translated on mitochondrial ribosomes and that the four smallest are formed in the cytoplasm. The results also underscore the advantages of using [³H]formate to identify the products of mitochondrial translation.     

Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system

Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.     

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)     

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.     

The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast.

Yeast cytochrome c oxidase contains three large subunits made in mitochondria and at least six smaller subunits made in the cytoplasm. There is evidence that the catalytic centers (heme a and copper) are associated with the mitochondrially-made subunits, but the role of the cytoplasmically-made subunits has remained open. Using a gene interruption technique, we have now constructed a Saccharomyces cerevisiae mutant which lacks the largest of the cytoplasmically-made subunits (subunit IV). This mutant is devoid of cyanide-sensitive respiration, the absorption spectrum of cytochrome aa3 and cytochrome c oxidase activity. It still contains the other cytochrome c oxidase subunits but these are not assembled into a stable complex. Active cytochrome c oxidase was restored to the mutant by introducing a plasmid-borne wild-type subunit IV gene; no restoration was seen with a gene carrying an internal deletion corresponding to amino acid residues 28-66 of the mature subunit. Subunit IV is thus necessary for proper assembly of cytochrome c oxidase.     

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.