J. Biochem. 90, 649-655 (1981)

The Subunit Composition of Pea Cytochrome c Oxidase1

Makoto MATSUOKA, Masayoshi MAESHIMA, and Tadashi ASAHI

Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University, Chikusa-ku, Nagoya, Aichi 464

Received for publication, March 18, 1981

Cytochrome c oxidase was purified from pea shoots in a form containing more than12 nmol of heme a per mg protein, but rapid inactivation took place during purifica-tion. On slab polyacrylamide concentration gradient gel electrophoresis of a par-tially purified preparation, there were three activity-bands corresponding to mainprotein bands. The activity-bands, as well as the most purified preparation, con-tained five polypeptides of different molecular weights (39,000, 33,000, 28,500,16,500, and 8,000-6,000) as shown by sodium dodecylsulfate-urea polyacrylamidegel electrophoresis. However, an immunoprecipitate from the partially purifiedpreparation with antibody against the most purified preparation contained twoadditional polypeptides with molecular weights of 13,000 and 10,000. Pea cyto-chrome c oxidase resembled the sweet potato enzyme with respect to immunologicalproperties and absorption spectra as well as the subunit composition. We proposethat higher plant cytochrome c oxidase is composed of five subunits of differentmolecular weights and is associated weakly with two low-molecular-weight poly-peptides in the mitochondrial inner membrane.

Cytochrome c oxidase [cytochrome c: O2 oxido-reductase, EC 1.9.3.1], the terminal oxidase in therespiratory chain tightly associated with the mito-chondrial inner membrane, has been isolated fromvarious eukaryotic cells including animal andfungal cells, and a great number of reports hasaccumulated concerning its subunit composition(see Ref. 1 for a review). Most of the reports haveshown that in eukaryotic cells the enzyme is com-posed of 6-8 subunits of different molecularweights (/): however, there is a report suggesting

1 This work was supported in part by grants (Nos.411308 and 448047) for Scientific Research from theMinistry of Education, Science and Culture of Japan.Abbreviation: SDS, sodium dodecylsulfate.

the existence of 12 different subunits in the en-zyme protein from mammalian cells (2). It hasbeen established that fungal cytochrome c oxidaseconsists of 7 subunits of different molecular weightsprobably in a stoichiometric molar ratio of one toone with one another (5). On the other hand,the enzymes from prokaryotes have been shown tocontain only 1-3 subunits (4-6).

Only one report is available, however, con-cerning the subunit composition of higher plantcytochrome c oxidase. Maeshima and Asahi (7)showed that the enzyme purified from sweet potatoroot tissue contained only 5 kinds of subunits andwas lacking in subunits of molecular weightsranging between 10,000 and 20,000 in spite of thewidespread occurrence of 2 or 3 subunits with

Vol. 90, No. 3, 1981 649

650 M. MATSUOKA, M. MAESHIMA, and T. ASAHf

such molecular weights in the enzymes from othereukaryotic cells. The purpose of the presentwork was to examine the subunit composition ofpea cytochrome c oxidase with purified enzymepreparations and an immunoprecipitate from acrude enzyme preparation, in order to determinewhether higher plant cytochrome c oxidase iscomposed of only 5 kinds of subunits, unlike theenzymes from other eukaryotes.

MATERIALS AND METHODS

Materials—Pea {Pisum sativwn cv. Alaska)seeds were purchased from Watanabe Seed Co.,Kogota, Miyagi, Japan. The seeds were surface-sterilized with 1 % NaOCl and germinated onvinyl nets placed just on the surface of deionizedwater at 25°C in the dark. The shoots of 6-day-old seedlings were used as the source for enzymepurification.

Cytochrome c (horse heart) was a product ofBoehringer Mannheim GmbH, and phosphatidyl-choline (egg yolk) was from Sigma Chemical Co.Molecular weight markers were purchased fromPharmacia Fine Chemical Co., and agarose andadjuvant were from DIFCO Laboratories Co.All other chemicals were of reagent grade.

Preparation of Submitochondrial Particles—A crude mitochondrial fraction was prepared bythe method of Bonner (8) with some modifications.Pea shoots obtained from 200 g of dry seeds werehomogenized with 200 ml of lOmin potassiumphosphate buffer, pH 7.2, containing 0.7 M man-nitol, 1.0 mM EDTA, and 2mM dithiothreitol ina blender. The homogenate was squeezed throughtwo layers of cheesecloth, and the filtrate wasadjusted to pH 7.2 with KOH. It was then cen-trifuged at 500 x g for 10 min, and the supernatantwas further centrifuged at 10,000 xg for 15 min.The precipitate was suspended in 10 mM potas-sium phosphate buffer, pH 7.2, containing 0.2 MKC1, and subjected twice to sonic oscillation at20 kHz for 20 s. The suspension was centrifugedat 100,000xg for 40min, and the precipitate(submitochondrial particles) was suspended in 12ml of 10 mM potassium phosphate buffer, pH 7.2.All the above procedures were carried out at0-4°C.

Purification of Cytochrome c Oxidase—Allthe following procedures for purification of pea

cytochrome c oxdase were performed at 0-4°C.Step 1: To the submitochondrial particle

suspension were added deoxycholate and anti-mycin A at final concentrations of 1 mg per mgprotein and 8.3 yt% per ml, respectively, and thesuspension was centrifuged at 100,000xg for 40-min. The pellet consisted of three layers; fluffytop, green middle and grey bottom layers. Themiddle layer, the bulk of the pellet, was suspendedin 10 mM potassium phosphate buffer, pH 7.2, to-a final volume of 18 ml. To the suspension wereadded solid KC1 and 20% Triton X-100 at finalconcentrations of 0.2 M and 2%, respectively.After being stirred for 20 min, the suspension wascentrifuged at 100,000xg for 40min, and thesupernatant was dialyzed against 10 mM potas-sium phosphate buffer, pH 7.5.

Step 2: The dialyzed preparation was ap-plied to a DEAE-cellulose (DE 52, Whatman}column (12x35 mm) preequilibrated with 10 mMpotassium phosphate buffer, pH 7.5, containing0.1% Triton X-100. The column was washed!first with the above buffer containing 0.4 % TritoaX-100 and then with the buffer containing 0.4%Triton X-100 and 0.07 M KC1. Finally, cyto-chrome c oxidase was eluted with the same buffercontaining 0.08% Triton X-100 and 0.17 M KC1.

Step 3: The eluted fraction was layered on15 ml of a linear sucrose density gradient rangingfrom 5 to 20% in 10 mM potassium phosphatebuffer, pH7.5, containing 0.1% Triton X-100 and0.1 M KC1. After centrifugation at 36,000 rpm(100,000 x g) for 16 h in a Hitachi RPS 40-T rotor,,greenish fractions were collected.

Step 4: The greenish solution was dialyzedagainst 10 mM potassium phosphate buffer, pH 7.5,.for 1 h, and applied to a DEAE-cellulose (DE 52,Whatman) column (7 x 30 mm), which had beeaequilibrated with the above buffer containing 0.1 %Triton X-100. The column was washed succes-sively with the following solutions; the same buffercontaining 0.1% Triton X-100, the buffer contain-ing 0.08% Triton X-100 and 0.08 M NaCl, and thebuffer containing 0.08% Triton X-100 and 0.16M:

NaCl. Cytochrome c oxidase was eluted with thefinal solution.

Sweet potato cytochrome c oxidase was iso-lated from root tissue as described previously (7).

Polyacrylamide Gel Electrophoresis—Disc elec-trophoresis in 10% polyacrylamide gels (acryl-

/ . Biochem.

SUBUNITS OF PEA CYTOCHROME c OXIDASE 651

amide : bisacrylamide=20 : 1) was performed inthe presence of 0.1 % SDS and 8 M urea followingthe general procedure of Swank and Munkres (9).Before being applied to the gels, samples were•stood in 2% SDS, 2% 2-mercaptoethanol, and 2 Murea at 70°C for 40 min. After electrophoresis thegels were stained for polypeptides with Coomassiebrilliant blue R, and scanned at 565 nm with aToyo Densitrol. Bovine serum albumin (67,000),•ovalbumin (43,000), chymotrypsinogen A (25,000),RNase A (13,700), and cytochrome c (12,300) wereused as markers for the estimation of molecular"weight.

Electrophoresis in slab gels with a linearpolyacrylamide concentration gradient ranging be-tween 3 and 15% (acrylamide : bisacrylamide=32 : 1) was carried out in the presence of 0.1%Triton X-100 in a cold room by the method ofNakashima and Makino (10). Each gel was•stained either for protein as described before orfor cytochrome c oxidase activity with the Nadimixture described by Keilin (11). Thyroglobulin<669,000), ferritin (440,000), catalase (232,000),Jactate dehydrogenase (140,000), and bovine serumalbumin (67,000) were used as molecular weightmarkers. The elution of protein from the gel was•done as described by Cleveland et al. (12). Gelslices were first incubated in 4% SDS containing4% 2-mercaptoethanol and 2 M urea at 30°C over-night, and then polypeptides in the slices wereeluted using an electrophoretic method. Theeluted polypeptides were precipitated by the addi-tion of trichloroacetic acid at 4°C overnight, theprecipitate was collected by centrifugation, washed•with ethylether, dissolved in the solution used fortreatment of gel slices, and subjected to SDS-ureapolyacrylamide gel electrophoresis.

Immunological Procedures—Antibody againstpea cytochrome c oxidase was raised in a rabbitby injecting three times a mixture of the finalpurified enzyme preparation (0.3 mg protein eachtime) and complete Freund's adjuvant at intervalsof one month, and the immunoglobulin G fractionwas prepared from the serum by ammonium sulfatefractionation and DEAE-cellulose column chro-matography. Antibody against sweet potato cyto-chrome c oxidase was prepared by a similar method(13).

Double immunodiffusion tests were carriedout according to the method of Ouchterlony (14).

The plates were composed of 0.8 % agarose, 0.4 %Triton X-100, 0.01 % NaN3, and 0.7% NaCl.

Analytical Measurements—Cytochrome c oxi-dase activity was assayed spectrophotometricallyat 25°C by following the oxidation of reducedcytochrome c at 550 nm in the presence of phos-phatidylcholine micelles as described previously(15). Absorption spectra were taken with aHitachi 200 recording spectrophotometer. Hemea was determined from the difference betweenabsorbances at 600 and 630 nm of dithionite-reduced cytochrome c oxidase provided that themillimolar extinction coefficient for the differencewas 16.5 (16). Protein was determined by themethod of Lowry et al. (17) with bovine serumalbumin as the standard after precipitation with10% trichloroacetic acid. SDS was added at afinal concentration of 0.5% for samples containingTriton X-100 (18).

RESULTS

Enzyme Purification—About 60% of proteinassociated with the submitochondrial particles,but not cytochrome c oxidase, was solubilized bydeoxycholate at a concentration of 1 mg per mgprotein. Almost all cytochrome c oxidase andheme a in the deoxycholate-treated submitochon-drial particles was solubilized by 2% Triton X-100,but not by other detergents such as deoxycholate(25 mg/mg protein) or cholate (50 mg/mg protein),in the presence of 0.2 M KC1.

The results of a typical purification from steps1 to 4 are summarized in Table I. The enzyme pro-tein was purified about 40-fold from the submito-chondrial particles with respect to heme a content.However, rapid inactivation occurred during thepurification: the activity per heme a declined toone fourth the original value during purificationstep 2, and complete inactivation took place duringsucrose density gradient centrifugation. Theinactivation seemed to be due to the presence ofthe rather high concentrations of Triton X-100.The exposure of the enzyme to Triton X-100 athigh concentrations for a long time caused inac-tivation, and none of the phospholipids testedhad the ability to reactivate the inactivated enzyme(Matsuoka, M. & Asahi, T., unpublished data).

The final preparation contained heme a at12.4 nmol per mg protein, which corresponded to

Vol. 90, No. 3, 1981

652 M. MATSUOKA, M. MAESHIMA, and T. ASAHI

TABLE I. Purification of pea cytochrome c oxidase.

Fraction Total protein Total heme a Heme a/protein Purific tion(mg) (nmol) (nmol/mg) (fold)

Submitochondrial fraction

Triton X-100 extract

Elute fraction after 1st DEAE-cellulose columnchromatography

Peak fraction after sucrose density gradientcentrifugation

Elute fraction after 2nd DEAE-cellulose columnchromatography

93.618.6

6.2

0.85

0.37

30.329.4

18.9

6.7

4.6

0.321.6

3.1

7.9

12.4

1

S 7

24.7

38.8

0.4

c

2: o , 2 -

A 439

/\A1 / j -Trace 2

1 / /',-i-Trace,

V /A /

\

400

.41 B

\ |<JA=0.1

\ 601

500 A 600/ \ Trace 3

J 601\

400 600500Wave length (n m)

Fig. 1. Absorption spectra of pea cytochrome c oxi-dase. The purified enzyme was diluted to a concen-tration of 0.38 mg protein/ml with 10 mM potassiumphosphate buffer (pH 7.2) containing 1.5% TritonX-100. (A) Absolute spectra of the air-oxidized (trace1) and sodium dithionite-reduced (trace 2) forms of theenzyme. Trace 3 is the spectrum of the reduced formbetween 480 and 640 nm expressed on an expanded scale.(B) Difference spectrum (reduced minus oxidized).

the contents of cytochrome c oxidase preparationspurified from other eukaryotic cells. The ab-sorption spectra of the oxidized and reducedforms of pea cytochrome c oxidase as well as thedifference spectrum between the reduced andoxidized forms were very similar to those of thesweet potato enzyme (4). The reduced formshowed a- and y- bands at 601 and 439 nm, respec-tively (Fig. 1A). There were maxima at 441 and601 nm in the difference spectrum (Fig. IB).

Immunological Properties—An active enzymepreparation (the preparation after purification step

Fig. 2. Ouchterlony double diffusion tests of cyto-chrome c oxidase from pea shoots and sweet potatoroots. (A) Immunological test of the final purifiedpreparation of pea cytochrome c oxidase. Center well:immunoglobulin to pea enzyme (40 ftg protein). Wells1, 2, 3, and 4: 15, 7.5, 3.75, and 1.9 fi& of the purifiedpea enzyme, respectively. (B) Comparison of peacytochrome c oxidase with the sweet potato one as toimmunological properties. Well 1: immunoglobulinto pea enzyme (13 fig protein). Well 2: immuno-globulin to sweet potato enzyme (50 fig protein).Well 3: partially purified pea enzyme (the preparationafter purification step 2, 13 ^g protein). Well 4: puri-fied sweet potato enzyme (4.7 fig).

J. Biochem.

SUBUNITS OF PEA CYTOCHROME c OXIDASE 653

2, 3.1 nmol heme a/mg protein) as well as thefinal one formed a single precipitin line againstantibody to the latter preparation in the Ouch-terlony double immunodiffusion test (Fig. 2). Theactive enzyme preparation also formed a singleprecipitin line against antibody to the sweet potatoenzyme, and vice versa, the purified sweet potatoenzyme did so against antibody to the pea enzyme(Fig. 2B). The precipitin lines were fused withone another, but a faint spur was observed betweenthe lines formed with the enzymes from the twosources.

Antibody to pea cytochrome c oxidase in-hibited the enzyme activity of the submitochondrialparticles by 60% (Fig. 3). It also inhibited theactivity of the solubilized enzyme to the samedegree. A similar result has been observed forthe sweet potato enzyme and its antibody (Mae-shima, M. & Asahi, T., unpublished data).

Polyacrylamide Concentration Gradient GelElectrophoresis—Figure 4 shows the distributionsof cytochrome c oxidase activity and protein onslab polyacrylamide concentration gradient gelelectrophoresis of the enzyme preparation afterpurification step 2 (4 nmol heme a/mg protein) inthe presence of Triton X-100. Three activity-

bands with respect to the Nadi reaction weredetected at positions corresponding to molecularweights of 670,000, 890,000, and 1,100,000, inaddition to a band at the top of the gradient gel.One of the bands (molecular weight, 670,000)appeared to be composed of two bands. Theactivity-bands corresponded to the main proteinbands.

Subunit Composition—5 main bands of poly-peptides with different molecular weights (I,39,000; II, 33,000; III, 28,500; IV, 16,500; and V,8,000-6,000: changes in polyacrylamide concen-tration had no significant effect on the values)were detected on SDS-urea polyacrylamide gelelectrophoresis of both the final enzyme prepara-tion (13 nmol heme a/mg protein) and an eluatefrom an activity-band in the slab gradient gel(corresponding to the position of a molecularweight of 890,000) (Fig. 5; traces A and B). Thesame results were obtained for eluates from theother activity-bands at positions of molecularweights of 890,000 and 1,100,000 in the slab gra-dient gel. There were minor bands at positionscorresponding to relatively high molecular weights,but the color intensity of the bands changed fromexperiment to experiment, suggesting that the

A B C

0 5 10Immunoglobulin G (mg / mg mitochondria)

Fig. 3. Inhibition of the activity of pea cytochrome coxidase by its antibody. Submitochondrial particlesfrom pea shoots (37 /*g protein) were incubated withthe indicated amounts of immunoglobulin to purifiedpea cytochrome c oxidase in a final volume of 200 ft\in the presence of 30 min potassium phosphate (pH 7.2)and 0.1% Triton X-100 at 25°C for 30 min, and thenassayed for cytochrome c oxidase activity.

Fig. 4. Slab electrophoresis ot a partially purifiedpreparation of pea cytochrome c oxidase in a poly-acrylamide concentration gradient (3-15%) gel con-taining 0.1% Triton X-100. Lane A: molecular weightmarkers (in the order from top to bottom, thyroglobulin,ferritin, catalase, lactate dehydrogenase, and bovineserum albumin). Lines B and C: pea cytochrome coxidase preparation after purification step 2 (heme acontent; 4 nmol/mg protein); lane B was stained forprotein, and lane C for activity.

Vol. 90, No. 3, 1981

654 M. MATSUOKA, M. MAESHIMA, and T. ASAHI

Migration

Fig. 5. Scans of SDS-urea polyacrylamide gels withthree different kinds of pea cytochrome c oxidase pre-parations. Trace A: the final purified enzyme pre-paration. Trace B: eluate from the activity-band cor-responding to the position of a molecular weight of890,000 in the slab gradient gel (see Fig. 4). Trace C:immunoprecipitate from the partially purified enzymepreparation after purification step 2 with anti-pea cyto-chrome c oxidase. Roman numerals denote the num-bers of subunits, whose apparent molecular weights are39,000 (I), 33,000 (II), 28,500 (III), 16,500 (IV), and8,000-6,000 (V). "a" and "b" indicate the polypep-tides with molecular weights of 13,000 and 10,000,respectively, detected only for the immunoprecipitate."L" and "H" indicate the light and heavy chains ofimmunoglobulin G, respectively.

bands were of aggregates of subunits.In the case of an immunoprecipitate from an

active enzyme preparation (the preparation afterpurification step 2), minor polypeptide bands were

consistently observed in positions correspondingto molecular weights of 13,000 and 10,000 (Fig.5: trace C).

DISCUSSION

Cytochrome c oxidase from animal and fungalcells has been reported to consist of 6-8 subunits(1), but the enzyme purified from sweet potatoroots is composed of only 5 subunits (7). Thepresent work showed that a purified, but inactive,preparation of pea cytochrome c oxidase also con-tained only 5 polypeptides of different molecularweights. The same results were obtained foractivity-bands with respect to the Nadi reactionon slab polyacrylamide concentration gradient gelelectrophoresis of an active preparation. There-fore, we deduce that active pea cytochrome coxidase is composed of only 5 subunits of differ-ent molecular weights. The molecular weights ofthe subunits of the pea enzyme resembled thoseof the sweet potato one: 39,000, 33,000, 28,500,16,500, and 8,000-6,000 for the former, and 39,000,33,500, 26,000, 20,000, and 5,700 for the latter.Both the enzymes also resembled each other inimmunological properties and absorption spectra.We propose that higher plant cytochrome c oxi-dase, which is active at least in the oxidation offerrocytochrome c with oxygen, is composed of 5different molecular weights.

However, an immunoprecipitate from a crudepreparation with anti-cytochrome c oxidase immu-noglobulin contained two other polypeptides withmolecular weights of 13,000 and 10,000 in addi-tion to the 5 polypeptides described above (Fig.5, trace C). A similar result has been observedfor sweet potato (Maeshima, M. & Asahi, T.,unpublished observation). Consequently, it seemsvery likely that higher plant cytochrome c oxidaseis associated weakly with two such polypeptidesin the mitochondrial inner membrane. The poly-peptides are not involved in the oxidation offerrocytochrome c. We have not yet studied thepossible role of these polypeptides, but there maybe a possibility of their participating in either theproton pump or the definite topological arrange-ment of the enzyme protein in the membrane.

Judging from the color intensity of bands ingels stained with Coomassie brilliant blue, the 5subunits seem to be present in a stoichiometric

J. Biochem.

SUBUNITS OF PEA CYTOCHROME c OX1DASE 655

ratio of 1 : 1 with one another in the enzymeprotein. Consequently, we assume the molecularweight of pea cytochrome c oxidase to be about125,000: the sum of the molecular weights of the5 subunits. One of the most purified preparationscontained 13.4 nmol heme a per mg protein thoughit had no activity, indicating that the molecularweight per heme a is about 72,500. Probably, theenzyme protein contains 2 heme a per moleculeand thus its molecular weight should be calculatedas 145,000 from the heme a content, consideringthat the molecular weight of the 5 subunits addup to about 125,000. On slab gradient gel elec-trophoresis, pea cytochrome c oxidase migratedto positions corresponding to molecular weightsof 670,000, 890,000, and 1,100,000 (Fig. 4). Wededuced that the enzyme migrated together witha Triton X-100 micelle in the electrophoresis.Cytochrome c oxidase has been reported to existin a form containing 4 heme a per unit molecule inTriton X-100 solution (19). Therefore, we pro-pose the three bands in the slab gradient gel tobe the dimer, tetramer, and hexamer of the enzymemolecule in the micelle. If so, the difference inthe molecular weight, 220,000, would reflect themolecular weight of the dimer of the enzymemolecule. Considering these calculations, we canimply that the molecular weight of pea cytochromec oxidase is within the range from 110,000 to150,000.

We express our thanks to Drs. Shio Makino and HiroshiNakashima, Department of Food Science and Tech-nology, of this faculty, for their helpful advice on slabpolyacrylamide concentration gradient gel electro-phoresis in the presence of Triton X-100.

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