The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

Topological studies of monomeric and dimeric cytochrome c oxidase and identification of the copper A site using a fluorescence probe

Beef heart cytochrome c oxidase was labeled at a single sulfhydryl group by treatment with 5 mM N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS) at pH 8.0 for 4 h. Sodium dodecyl sulfate gel electrophoresis revealed that the enzyme was exclusively labeled at subunit III, presumably at Cys-115. The high affinity phase of the electron transfer reaction with horse cytochrome c was not affected by acetylamidoethyl-1-aminonaphthalene-5-sulfonate (AEDANS) labeling. Addition of horse cytochrome c to dimeric AEDANS-cytochrome c oxidase resulted in a 55% decrease in the AEDANS fluorescence due to the formation of a 1:1 complex between the two proteins. Forster energy transfer calculations indicated that the distance from the AEDANS label on subunit III to the heme group of cytochrome c was in the range 26-40 A. In contrast to the results with the dimeric enzyme, the fluorescence of monomeric AEDANS-cytochrome c oxidase was not quenched at all by binding horse heart cytochrome c, indicating that the AEDANS label on subunit III was at least 54 A from the heme group of cytochrome c. These results support a model in which the lysines surrounding the heme crevice of cytochrome c interact with carboxylates on subunit II of one monomer of the cytochrome c oxidase dimer and the back of the molecule is close to subunit III on the other monomer. In order to identify the cysteine residues that ligand copper A, a new procedure was developed to specifically remove copper A from cytochrome c oxidase by incubation with 2-mercaptoethanol followed by gel chromatography. Treatment of the copper A-depleted cytochrome c oxidase preparation with 1,5-I-AEDANS resulted in labeling sulfhydryl groups on subunit II as well as on subunit III. No additional subunits were labeled. This result indicates that the copper A binding site is located at cysteines 196 and/or 200 of subunit II and that removal of copper A exposes these residues for labeling by 1,5-I-AEDANS. Alternative copper A depletion methods involving incubation with bathocuproine sulfonate (Weintraub, S.T., and Wharton, D.C. (1981) J. Biol. Chem. 256, 1669-1676) or p-(hydroxymercuri)benzoate (Li, P.M., Gelles, J., Chan, S.I., Sullivan, R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095) were also investigated. Treatment of these preparations with 1,5-I-AEDANS resulted in labeling cysteine residues on subunits II and III. However, additional sulfhydryl residues on other subunits were also labeled, preventing a definitive assignment of the location of copper A using these depletion procedures.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

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

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

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.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

Location of heme a on subunits I and II and copper on subunit II of cytochrome c oxidase

A systemic study has been made of copper and heme a binding to subunits of beef heart cytochrome c oxidase. Copper and heme a were readily mobilized by ionic detergents, high ionic strengths, temperatures above 0 degrees C, thiol compounds, and gel-bound peroxides and free radicals when the subunits of the oxidase were dissociated from one another during polyacrylamide gel electrophoresis. Most subunits showed some affinity for heme a and copper under these conditions. However, in the presence of specific mixtures of ionic and nonionic detergents (e.g. 0.1% sodium dodecyl sulfate, 0.025% Triton X-100) at temperatures below 0 degrees C and in buffers of low ionic strength using 10 to 12% polyacrylamide gels preelectrophoresed for 3 days with thioglycolate, about 90% of the Cu was found on subunit II (Mr = 24,100), and heme a was found in equal amounts of subunits I (Mr = 35,800) and II. The oxidized-reduced and reduced-CO absorption spectra of these subunits resembled those of cytochrome c oxidase. It appears probable that in the native enzyme, subunit I contains heme a and subunit II contains copper and heme a. A relationship of mammalian cytochrome c oxidase to the two-subunit microbial cytochrome oxidase systems appears to exist.     

Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species.

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.     

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.     

The localization of tightly bound cardiolipin in cytochrome oxidase

One to two molecules of tightly bound cardiolipin are associated with resolved fractions of cytochrome oxidase containing subunits I to III or I to IV. Large scale isolation of subunits I to IV indicates the presence of approximately 0.5 molecule of cardiolipin per molecule of subunit I. Lipoprotein staining of sodium dodecyl sulfate/urea/acrylamide gels of cytochrome oxidase support the findings that subunit I is a lipoprotein. The resistance of this tightly bound cardiolipin to organic solvent extraction suggests a specific association of some tenacity with the protein.     

The isolation of bovine-heart cytochrome c oxidase subunits. Dependence on phospholipid and cholate-content.

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate. The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated. Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS. Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts. The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.     

Expression of cyoA and cyoB demonstrates that the CO-binding heme component of the Escherichia coli cytochrome o complex is in subunit I

The cytochrome o complex of the Escherichia coli aerobic respiratory chain is a ubiquinol oxidase. The enzyme consists of at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and contains two heme b prosthetic groups (b555 and b562) plus copper. The sequence of the cyo operon, encoding the subunits of the oxidase, reveals five open reading frames, cyoABCDE. This paper describes results obtained by expressing independently cyoA and cyoB in the absence of the other subunits of the complex. Polyclonal antibodies which react with subunits I and II of the purified oxidase demonstrate that cyoA and cyoB correspond to subunit II and subunit I, respectively, of the complex. These subunits are stably inserted into the membrane when expressed. Furthermore, expression of cyoB (subunit I) results in elevated heme levels in the membrane. Reduced-minus-oxidized spectra suggest that the cytochrome b555 component is present but that the cytochrome b562 component is not. This heme component is shown to bind to CO, as it does in the intact enzyme. Hence, subunit I alone is sufficient for the assembly of the stable CO-binding heme component of this oxidase.     

Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene

Cytochrome c oxidase was treated with 1,5-difluoro-2,4-dinitrobenzene at molar ratios (DFDNB:oxidase) varying from 5 to 625. At the lowest ratio, there was virtually no effect of the probe on oxidase activity or on migration of oxidase subunits on sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis. At ratios of 25 and greater, there was loss of oxidase activity and a change of the pattern of subunit migration on sodium dodecyl sulfate electrophoresis. (i) Activity loss was probably a result of severely perturbing the cytochrome c binding site since oxidase activity with a low molecular weight reductant (N,N,N',N'-tetramethylphenylenediamine) was unaltered. Also unaltered were the oxidized, reduced, and carbon monoxide binding spectra of the treated oxidase. (ii) The staining pattern on sodium dodecyl sulfate electrophoresis showed that subunits III and VI disappeared from their normal positions on the gel. A new band of higher molecular weight accompanied their loss from the gel indicating that the two subunits were being cross-linked. Subunits III and VI are thus shown to have two reactive groups within 4.8 A (1 A = 0.1 nm) of one another. This proximity has not been detected with other probes that react with the same groups.     

The role of subunit III in bovine cytochrome c oxidase. Comparison between native, subunit III-depleted and Paracoccus denitrificans enzymes

In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82 000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1-2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation.     

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.     

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.     

The nature of zinc in cytochrome c oxidase.

The zinc ion in bovine heart cytochrome c oxidase can be completely depleted from the enzyme with mercuric chloride without denaturing the protein. The metal atom stoichiometry of 5Cu/4Fe/0Zn/2Mg obtained for the enzyme following HgCl2 treatment indicates that this depletion is highly selective. Zinc depletion exposes one cysteine on subunit VIa and one cysteine on subunit VIb for N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine (1,5-I-AEDANS) labelling, suggesting that the zinc plays a structural role in the protein by providing a bridge between these two subunits. Although the treatment of cytochrome c oxidase with mercuric chloride inhibits the steady-state activity of the enzyme, subsequent removal of the Hg2+ bound to cysteine residues by 1,5-I-AEDANS significantly reverses the inhibition. This latter result indicates that the removal of the zinc itself does not alter the steady-state activity of the enzyme.     

Large scale isolation and properties of subunits from bovine heart cytochrome oxidase

The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitable for amino acid sequence studies. The preparation of subunits III, IV, V, VI, and VII for sequence determination can be achieved without employing sodium dodecyl sulfate. The method presented essentially involves pyridine extraction, pH fractionation, ammonium sulfate fractionation, and various types of column chromatography. However, subunits I and II can be prepared only in the presence of sodium dodecyl sulfate by molecular sieve chromatography; subunit III can also be isolated in this manner. The separation of subunits is found to be hindered by phospholipids associated with the enzyme and therefore the phospholipid-depleted preparation is used as the starting material. The molecular weights of subunits I, II, III, IV, V, VI, and VII are 40,000, 21,000, 14,800, 13,500, 11,600, 9,500, and 7,600, respectively. These values are based on the results of the conventional Weber and Osborn method of gel electrophoresis in the presence of sodium dodecyl sulfate. The amino termini of subunits I and II have been determined as N-formylmethionine, and those of subunits III, IV, V, VI, and VII are alanine, alanine, serine, alanine, and an N-acetyl-blocked residue, respectively. The carboxyl termini for subunits I to VII are lysine, leucine, lysine, histidine, valine, isoleucine, and valine, respectively. The complete amino acid sequence of some subunits has been published and that of other subunits will be reported elsewhere in collaboration with the Amino Acid Sequence Group of Cytochrome Oxidase at the University of Hawaii.     

An active cytochrome c oxidase depleted of subunit III prepared by covalent chromatography on yeast cytochrome c

A covalent chromatography technique is described for the preparation of an active cytochrome c oxidase from bovine heart devoid of subunit III. Yeast cytochrome c is immobilized on a Sepharose 4B gel, its cysteine 107 activated and reacted with the oxidase. Elution with Triton X-100 releases an oxidase devoid of subunit III, which is recovered after elution with β-mercaptoethanol.