Isolation, subunit composition, and site of synthesis of human cytochrome c oxidase

Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), the terminal oxidase of the respiratory chain in eucaryotic cells, has been purified from human placenta mitochondria. Seven polypeptides have been identified reproducibly by high-resolution electrophoresis of the enzyme complex through sodium dodecyl sulfate (Na-DodSO4)--urea polyacrylamide gels; these correspond closely in size to the subunits of beef heart cytochrome c oxidase. When HeLa cells, grown in suspension culture, were pulse-labeled with [35S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis and chased with an excess of unlabeled methionine in the absence of the drug, the mitochondrially synthesized polypeptides were resolved into at least 17 components by NaDodSO4--urea polyacrylamide gel electrophoresis. After labeled HeLa mitochondria were mixed with human placenta mitochondria and the cytochrome c oxidase was isolated, three of the labeled components were found to copurify with the three largest subunits of the complex. We conclude that human cytochrome c oxidase contains seven subunits, the three largest of which are synthesized on mitochondrial ribosomes, while the other four are synthesized in the cytoplasm.     

Immunoprecipitation of a cytoplasmic precursor of rat-liver cytochrome oxidase.

A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.     

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.     

Synthesis of the proteins of complex III of the mitochondrial respiratory chain in heme-deficient cells

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5-aminolevulinic acid synthase and, hence, devoid of heme. The two 'core' proteins, apocytochrome b and the iron-sulfur protein, were observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against complex III and the sensitive immuno-transfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1. By contrast, when mitochondria from the heme-deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

Biochemical characterization of the OXI mutants of the yeast Saccharomyces cerevisiae

OXI mutants in Saccharomyces cerevisiae lack a functional cytochrome c oxidase. Wild type and OXI mutants were grown in the presence of radioactive delta-amino[14C]levulinic acid, a precursor of porphyrin and heme, and [3H]mevalonic acid, a precursor of the alkyl side-chain of heme a. SDS polyacrylamide gel electrophoresis of the delipidated mitochondria showed that delta-amino[14C]levulinic acid was distributed into three bands migrating in the regions of Mr 28 000, 13 500, and 10 000, while [3H]mevalonic acid was found in a single band with apparent Mr of 10 000. The immunoprecipitates obtained by incubating the solubilized mitochondria of any OXI mutant with antibodies against cytochrome c oxidase, showed, after delipidation, a high specific radioactivity due to delta-amino[14C]levulinic acid and [3H]mevalonic acid. This suggested that a prophyrin a was present in all these OXI mutants. HCl fractionation confirmed the presence of porphyrin a in the apooxidase of these mutants. Atomic absorption spectra of the immunoprecipitate of cytochrome c oxidase showed that copper was not detectable in the mutant OXI IIIa which lacked subunit 1, but was present in the mutant OXI IIIb, which exhibited a minor alteration in the electrophoretic mobility of subunit 1. In OXI I and II mutants there was a 50% reduction in the amount of copper in the immunoprecipitated cytochrome c oxidase. These observations may be interpretable as follows: (1) alterations in polypeptide biosynthesis due to the OXI mutations lead to an improper configuration of cytochrome c oxidase, so that ferrochelatase cannot transfer iron into porphyrin a; (2) subunit I is the binding site for copper, but the mutations in subunits II and III alter the binding site of one of the two copper atoms in subunit I.     

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa₃ terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n-dodecyl-β-d-maltoside and purified by ion-exchange chromatography using poly-L-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)⁻¹ and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a ₃, furthermore oxygen consumption was found to be sensitive to cyanide.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.     

Purification and characterization of the cytochrome oxidase from alkalophilic Bacillus firmus RAB

A cytochrome oxidase was purified 52-fold from membranes of alkalophilic Bacillus firmus RAB by extraction with Triton X-100, ion-exchange and hydroxyapatite chromatography, and gel filtration. On denaturing gels, the purified enzyme dissociated into two subunits of 56,000 and 40,000 Mr as well as a cytochrome c with an Mr of approximately 14,000. Heme contents calculated for an enzyme with a molecular weight of 110,000 were found to be 2 mol of heme a and 1 mol of heme c per mol of cytochrome oxidase; approximately 2 mol of copper per mol of purified enzyme was also found. Enzyme activity was observed in assays using reduced yeast or horse heart cytochrome c. Activity of the purified enzyme was optimal at pH 6.0 and in the presence of added lipids. Impure, membrane-associated activity exhibited a broader pH range for optimal activity extending to alkaline values.     

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.     

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.     

Hydrophobic interactions of cytochrome c oxidase. Application to the purification of the enzyme from rat liver mitochondria.

The binding of rat liver cytochrome c oxidase to phenyl-Sepharose and various alkyl and omega-aminoalkyl agarose gels has been studied. Deoxycholate-solubilized cytochrome c oxidase was tightly bound to hexyl, octyl, omega-aminohexyl, omega-aminooctyl agarose as well as to phenyl-Sepharose. This hydrophobic interaction was used for the purification of cytochrome c oxidase. The enzyme which was eluted from phenyl-Sepharose was devoid of NADH (NADPH)-acceptor reductase activities. The heme a content was 15.4 nmol per mg of protein. The purified enzyme was resolved into seven polypeptides upon polyacrylamide gel electrophoresis in sodium dodecylsulfate with molecular weights of 40,000, 23,200, 21,500, 14,500, 12,600, 8900, and 4900. Antibodies raised in rabbits against the pure enzyme did not cross-react with cytochrome c oxidases from either beef heart or yeast mitochondria. Cytochrome c oxidase bound to octyl-Sepharose or phenyl-Sepharose exhibited a very low catalytic activity. The possible modes of interaction of cytochrome c oxidase with the hydrophobic ligands are discussed.     

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus)

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.     

Structure and orientation of cytochrome c oxidase in crystalline membranes. Studies by electron microscopy and by labeling with subunit-specific antibodies.

The structure and the orientation of cytochrome c oxidase molecules in crystalline cytochrome c oxidase membranes (Vanderkooi, G., Senior, A.E., Capaldi, R.A., and Hayashi, H. (1972) Biochim. Biophys. Acta 274, 38-48) were studied by image analysis of electron micrographs and by reacting the crystalline preparations with immune gamma-globulins against individual cytochrome c oxidase subunits. Binding of gamma-globulins to the membranes was detected by the following two methods: (a) electrophoretic identification of gamma-globulin polypeptides in the washed membranes; (b) electron microscopic examination of the negatively stained membranes. The membranes bound immune gamma-globulins against subunit IV (which faces the matrix side in intact mitochondria) but failed to bind immune gamma-globulins against subunits II + III (which face the outer side of the inner membrane in intact mitochondria). In contrast, solubilized cytochrome c oxidase bound either of the two immune gamma-globulins. All cytochrome c oxidase molecules in the crystalline membranes are thus asymmetrically arranged so that subunit IV faces outward and subunits II + III face toward the interior. This orientation is opposite to that found with intact mitochondria. The data also suggest that the crystalline membranes form closed vesicles which are impermeable to externally added gamma-globulins.     

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.     

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.     

Mitochondrial respiratory chain of Tetrahymena pyriformis: the properties of submitochondrial particles and the soluble b and c type pigments.

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 degree C there are two b-type pigments with half-reduction potentials of --0.04 and --0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a2 with Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g =6, and a g=2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed. Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD of 0.8.10(-6) M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g=3.28. A pigment with an alpha absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.     

No unique mitochondrial translation products in respiratory chain-linked NADH dehydrogenase

Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[³⁵S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase.     

Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides

With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species.     

Proteolytic precursor processing in the biosynthesis of mitochondria

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.