Studies on the electron-transfer system LX. Molecular weights of some components of the electron transfer chain in beef-heart mitochondria

mtxmLum.,, ~T BtoPii~-smA AcIA

i

~u~ 65~3o STUDIES ON T H E ELECTRON-TRANSFER SYSTEM

LX, ,VOLECULAR W E I G H ' I S OF SOME COMPONENTS OF T H E ELECTRON TRANSFER CHAIN 1N BEEF-HEART MITOCHG,"."DRIA

A L E X A N D E R rZAG¢ILOFF. P A U L I N E C. YANG, D A V I D C W H A R T O N S. R 1I}:SKE tnst~mt,: fi~r t~2~z3+t~• ~,?¢sca~vh, U~,;zersity qt" tVisco~sb~. Madison, W i s e ( U . S . : I .) (Rec'eiv~xl Jlliy 7th. 19r.,41

~D

JOHN

SUMMARY

The m~qev~lar weights of two enzyme complexes purified :tom beef-heart mitochondria have l~een determined by the light-scattering tech~:iqae. The enzymes ~tudied were c~umzyme QI-L~cyt~×:hrome c reducta~ (Complex II! ~ and cvtoehrome c oxidase (cyt(~:hnmte c:O. oxidort~uctase, EC r.9.3.I) (Complex IV). 1 [~e weight average molecular weight of Complex I I I from tight-scattering data agrees, losel3 with the minimal nmkvular weight estimated from the c3~odmmle c, content. In the case of cyttu:hr(~me oxidase, however, the molt~:ular weight was about ~wo times larger than '~he minimM moltx-uktr weight based on the cytoehrome a content. This observation ,,upI~fts tile view that tlmre are tw~ heine a prosthetic g r ) l p s per molecule of cyttwhrome oxidase.

I N rRODUC I'ION

Past work in this laboratory 1°'~ has established the exi-, ence of four separable enzyme complexes in btx, f-heart mitochondria which together ~mplement the sequential transfer of electrons from D P N H and suecinic acid to oxy ,'t~ Alqmugh the comtx~sition and enzymic pmi×,rties of each complex have been .....11chaxacterized, physieM measurements such as molecular weights have not been t : "tied out in any detail. "File m~flecular weights whk-h have been reported for the c.~l ,,plexes were minimal values calculated on tim basis of the content of s~nne pros',' ::i, group (e.g. heine or ilavin) characteristic of tile complex. rhe d-.,Ugatory u ~ of bile ~.lts or detergents to distx ~,~ particulate proteins presents serious problems in the interpretation of data ob':~ ned b7 means of the analytical ultracenn'~ifilge, We have therefore applied light-s atteril>g techniques as an altema~:ive approad~ ~o the determinatim', of tile molecula,: ~'m~ht cf these protein compk~xcs~ l a this communication we reD~rt stone studies o~ ~hc molecular weights ,of two of the comple×es derived from the electron-transfer ::hain, The eomNexes chosen were ct~nzyme QHe
2

A. TZAGOLOFF ~ a 2 .

oxidase (cytochrome c:02 oxidoreductase, EC 1.9.3.1 ) (Complex IV). The ultracentrifugal patterns indicated that both of these complexes were sufficiently monodisperse for light-scattering studies. METHODS

The purification of Complex I i I was based on the method described by HATEFI

et alp as modified by RIESKE el, al. 6. Complex IV was prepared by the method of GRIFF[THS Arid ~THARTON4.Taurocholate and taurodeoxycholate were synthesized from cholate and deoxycholate, respectivelyL

Anal~ "ical ultracentrifuge Sedimentation coefficients were determined with a Spinco Model E ultracentrifuge equipped with a phase plate schlieren diaphragm. Sedimentation was '~,:rformed with the rotor temperature controlled within the range 7-1o °. The sedi~:ntation coefficients were corrected for the rotor temperature, solvent density, and . 'Avent viscosity to that of water at 20 °. The partial specific volumes were assumed to be 0.74 for both complexes.

Refra~ "re index Refractive index measurements were made with a Brice-Phoenix differential refractometer. The enzymes were equilibrated with the solvent system by prolonged dialysis (24-48 h). The solvent system used with Complex I I I was: 0.66 M sucrost, o.o5 M Tris-HC1 (pH 8.o), o . I /O/o taurocholate and ammonium sulfate (5°/o saturatio: 0. The solvent system used with Complex IV was: o.25 M sucrose, o.o5 M phosphate buffer (pH 7-5), o.1% taurodeoxycholate and ammonium sulfate (5% saturatic,,3. Refractive indices were determined at 3-5 °.

Light-scatteriv g Light-scattering was measured with a Brice-Phoenix photometer. An ap7: priate amount of the concentrated enzyme (2o-3o mg of protein per ml) was dilm..d with the specified solvent to a final volume of 3 ml. The soh'ent used for diluting Complex I I i was: o.66 M sucrose, o.o5 M Tris-HC1 (pH 8.o), o.33% taurocholate*. The solvent for Complex IV was: 0.25 M sucrose, o.o5 M phosphate buffer (pH 7.5) and o.33% taurodeoxycholate. Upon dilution the solution was immediately transferred to a I-cm square micro cell and readings of light scattered at 9 °0 were taken at intervals of 15 see over a period of 2-3 min. A zero angle reading was obtained after 3 rain ;this value did not change over the 3-rain period. Since scattering at 9 °0 increased during this time period, the absolute turbidity of the solution was taken as the value obtained by extrapolating to zero time (cf. Fig. 2). All data were corrected for lightscattering contributed b y the solvent systam. Both enzyme and solvent solutions were clarified by filtration through a millipore filter (o.45/,). Light-scattering was measured at 3-5 °. * In a few experiments light-scattering was measured in a solvent system corresponding to that used for refractive index rnez~urements. NO differences were found to exist in the lightscattering properties of the enzyme in the two solvents.

Bioahim. Biophys. Aaa, 96 (I965) 1-8

MOLECULA~ VCEIGHTS OF COMPLEXES I I I AND I V

3

Analytical m ~thods The cytochrome a content of cytochrome oxidase was determined from the reduced spectrum, the value I6. 5 cm -1 mole -x being used as the extinction coefficient fo:" tl,e difference in absorbancy between 605 and 63o m~ iref. 8). The cytochrome c1 content of Con~plex I I I was determined by "he method of ZAUGG AND RIESKE9. Total phos#lorus was assayed by the method of CrIEx et al. 1°. The lipid content was calculated fix,m the phosphorus value u. Ly'coclaronte oxidase activity was assayed by the spectrophotometric m.~thod Of SMITH 12.

Proteins were determined by the method of LOWRy'et al. is axed by the biuret method of GORNALLet al. x~. The two meth~ds agreed withi~a 5%, RESULTS

Analytical ultracentrifuge The arv,dyticM ultracentrifuge patterns of Complexes,: H I and IV are shown in Fig. Ia and Fig. Ib, respectively. Complex I I I sedimented a~s a single color-.associated boundary, whereas the slow moving, colorless sedimenting boundary corresponded

Fig. la. S e d i m e n t a t i o n p a t t e r n of Complex l I I . The e n z y m e was dialyzed against the following solvent: o.66 M sucrose, c,.o 5 M Tris-HC1 (pH 8.0), o.I~o taurocholate :,nd a m m o n i u m sulfate (5 o~) saturation). The picture was t a k e n 174 rain after t h e rotor reached 59 780 rcv./miu. The rotor t e m p e r a t u r e was 7.5 °. b. Sedimentation p a t t e r n of Complex I V. The e n :yme was (liaiyzod a g a i n s t the following solvent: 0.25 M sucrose, o.o5 M p h o s p h a t e buffer tprt 7.5), o.I°'~ taurodeo×ycholate a n d a m m o n i u m sulfate (.~-°e;osaturation). The picture was t~ken 64 min after the rotor re.ached 59 780 rev./min. The rotor t e m p e r a t u r e was Io °.

to a detergent micelle. The sedimentation coefficient of Cor~ Aex I I I when corrected for water at 2o ° is lO.2 S, a value which is 4-5 times larger t.'~an the apparent sedimentation coefficient at 7.5 °. Complex IV suspended in the sucrose-detergent buffe. ,ystem sedimented as a major colored boundary which had an s2o.,v of 12 S. The e. ~mined preparation exhibited two other minor protein boundaries as well as a de ergent boundary. The contaminating protein accounted for about 5 ~ of the tot;~.l :~rotein

Refractive index Measurements of refractive index were made at 587, 54' >~'~nd436 m#. The values presented in Table I are averages obtained from measuremel~t ~made at three or four protein concentrations. Although the values of refractive ~:,,dcx iucrements were Biochim. E." ~phys. Actor, 96 (t965) t.-8

A. TZAGOLOFF el~ al.

.

TABLE I R~FR^eTIvB INDEX ISCRE~mNTS (C I~ g/ml) Complex

III IV

546 m#

587 mix

U.ncorrected Corrected for lipid

Uncorrected

Corrected for lipid

Unc,orrected

Corrected for lipid

o.zi 7 o.269

o.218 0.270

o.I89 o,z:ol

o.~ 3 --

o.I94 --

o.z88 o.zoI

436 m/t

higher t h a n t h o s e usually f o u n d for proteins the5" fell w i t h i n t h e c u s t o m a r y r a n g e w h e n c o r r e c t e d for t h e c o n t r i b u t i o n m a d e b y the associated lip;d*. Light-scattering B o t h Complexes I I I a n d I V showed a t e n d e n c y to a g g r e g a t e w h e n d i l u t e d at c e ,centrations below 5 m g of protein per ml. T h e aggregation, as reflected b y lights, a~tering at 9 o°, was linear w i t h time. Fig. 2 shows the increase in t h e s c a t t e r i n g t~.~ R~o/Roo o f Complex i l i in the presence of 0 . 3 3 % t a u r o c h o l a t e . W i t h lower c o n c e n t r a t i o n s of taurocholate, t h e t u r b i d i t y increased faster; however, t h e values

/°

0.07 0 06 o o

~ _._._ . _ _ , ~

o.o4-/'° o

~,58

. . . . .

/

005

.3.5

_4~5

. . . . . }~

0~03

2.3

0.02 0.01L--'--"

30

--"

~

'

60 90 120 fltne (sec)

,,

.J.2

150

Fig. 2. Increase in the scattering ratio of Complex 111 with time. Complex Ill was diluted with a soh,ent system containing o.33 % tanrocholate (O--O) to the indicated protein concentrations. When no bile acid was present in the diluting medium the scltttering ratio increased more rapidly

(o--o). *

formula:

The specific refractive index increment of the protein may be calculated by the following dNp

dNp+ l

de.

de.

dNl

(I,~

TCq~\i-,/

dNp dC-~ = specific refractive index incre~mnt of protein, dNp+~ -~

observed specific refractive index

dNl increment based on the concentration of protein, d ~ l = specific refractive index increment of the protein-bound lipid (this vMue is assumed to be about oA 7 according to unpublished studies of WASSERMAN AIqD F L E I S C H E R ) .

Biochim. Biophys. Acta, 96 (r965) i-8

MOLECULAR ~.~rEIGHTSOF COMPLEXES I I I AND I V

5

o f a b s o l u t e t :,rbidity e x t r a p o l a t e d to zero t i m e were t h e s a t , e, e v e n w h e n t h e bile salt was o m i t t e d f r o m t h e d i l u t i o n m e d i u m . A t a p r o t e i n c o n c e n t r a t i o n of 5 m g p e r m l or lower, t h e p e r c e n t transmission a t 587 a n d 54 ° n~u was g r e a t e r t h a n 37- T h e s c a t t e r i n g ratios o b t a i n e d at these w a v e lengths were, therefore, used d i r e c t l y w i t h o u t a n y correction for color. A t 436 m F b o t h e n z y m e s a b s o r b e d s t r o n g l y a n d s c a t t e r i n g was m e a s u r e d o n l y at c o n c e n t r a t i o n s below 2 m g o f p r o t e i n p e r mL W h e n the per c e n t t r a n s m i s s i o n at this w a v e l e n g t h was lower t h a t , 37, suitaMe corrections were applied to the s c a t ~ d n g ratios (see B r a c e et alo15).

W e i g h t a v e r a g e m o l e c u l a r w e i g h t s o f t h e t w o c o m p l e x e s were calculated b y using a r e f r a c t i v e index i n c r e m e n t b a s e d on the t o t a l weight ~f the e n z y m e (protein p l u s lipid). T h e inolecular weight e f t h e p r o t e i n was t h e n c;I~tained b y s u b t r a c t i n g the e x p e r i m e n t a l l y d e t e r m i n e d a m o u n t o f lipid b o u n d to the protein.

I.ol

L0 ~o

O,B,~

~o

N

~,, 0.6 LJL~

•

~'

0.4.

I,q

~

o

"'o . . . . .

o

12 , ,e

°

--

"

"

•

~ 0-6

~

0.8

0.4

0.[3

--o--~

0.2

Z

1.0

•

~

~ .a~o.._. o. . . . . . . .

0.8

~* ~ ~ o "

o---.---if-

0.2 q

2

3 * conch {m~ / roll.

5

t

2 3 con~(rng / ml )

.~,

4

Fig. 3, Light-scattering data for Complex III plotted to show the molecular weight ( O - - O ) and h'aso/Ri~,o ( H ) dependence on concentration of enzyme. Fig. 4. Light-scattering data for Complex IV plotted to show the molecular ::Cght (C- -[)) and Raso[/~i~° ( H ) dependence on concentration of enzyme. The d e p e n d e n c e of H c / v o n c o n c e n t r a t i o n of e n z y m e is p l o t t e d in Figs, 3 and 4 t'.r Complexes I I I and IV, respectively. The weight a v e r a g e m o l e c v i a r weights were d e t e r m i n e d from t h e reciprocals of the i n t e r c e p t s at zero conc-en~ration. U n d e r the condition.: used in this s t u d y the molecular w..fights a p p e a r e d ~ ~ be i n d e p e n d e n t of FABLE II W~-,TG}IT A V F R A G E

::oreblex

WEIGHTS

OF COMPLEXES

Molecular u,ights uncorrected mg lipid for lipid per mg ........................................................ protein 546 mtt 587 rot* 436 mtz

¢II I. 303000 2. 3z7 ooo IV t. 2, 3. 4.

MOLECULAR

3200o0 333oo0 . . . .

296 ooo 32I ooo 303 OOO --312 OOO -266 oo0 .

.~----.

.

o.158 . . 0.336 0.336 0.338 .

IIi AND IV

Molec,dar weights corrected # r lipid ...................................... 546 ml2 587 mp 436 ~rn~

26200o . .

277000 . .

288o(,0, . .

220 ooo

240 ooo ~ --

--~ --

226 ooo

234 ooo . .

Cylochrome content (mltmoles/mg 15~u;~in) ...............

Minimal molecular weight on basis of cytochrome

¢~

Ct

CI o r

3.74

268000

.

. 7.4 8. 3 8.0 8. 7

135 000 t22 o00 I25 0o0

11,50~O

Biochim. Biopkys. Aela, 96 (I965) x-8

6

A. TZAGOLOFF ~ a / .

protein concentration. The scattering ratios R4so/R135o for several concentrations are shown also in I~Jgs. 3 and 4. Neither enzyme demonstrated significant dissymmetry of ~:attering at these two angles~ Table II summarizes the molecular weights of Complex I I I and of Complex IV. The weight average molecular weight of Complex I I I is about 300 ooo, in good agreem~nt with the minimal molecular weight based on the cytochrome c1 content. The averaged molecular weight found for four preparations of Complex IV was 290 ooo. When corrected for lipid, :the weight average molecular weight of the protein was 23o ooo, I n further experiments it was found that the apparent size of Complex IV was lowered to about ioo ooo in the presence of 4 or 6 M urea. Higher concentrations of biie salt, t h e presence of sodium dodecyI sulfate, or alkaline p H had no apparent effect on the molecular weight of Complex IV.

DI%CUSSION

Methods for the isolation in highly purified form of enzyme complexes of the electron-transfer chain have made possible studies of certain physical properties of these enzymes. The observation that two of the complexes, namely Complex I I I and Complex IV, could be obtained as nearly homogeneous proteins prompted us to examine their molecular weights by tile ligl:t-scattering technique. Ill this study it was of interest to determine whether the molecvlar weights obtained hy a physical method corresponded with the molecular weights estimated from chemical composition. With Complex III, the close agreement found between tile experinaental and calculated molecular weights supports the previous view3,", ~6that this enzyme contains two molecules of cytochrome b, one molecule of non-home iron protein, and one molecule of cytochrome q. The average molecular weight of Complex IV was 230 ooo. Since the average minimal molecular weight, based on its content of cytochrome a, was about I2O ooo we conclude that there are probably two home prosthetic gronps per molecule of Complex IV. TAKE~fORi etal. ~7 have reported that their preparation of Complex 1V has a molecular weight of 53o ooo. Since these authors did not specify the lipid content of their preparation a direct comparison between their value and ours cannot be made. If we assume a comparable amount of lipid in the two preparations, our value remains significantly lower. Past studies on the stoichiometry of the components of the electron-transfer chain in beef-heart mitochondria~';, t8 have shown that there are six molecules ~Jf cytochrome a per unit chain. It was, therefore, proposed that Complex IV exists as a hexamer. The present study indicates that the unit of Complex IV is built up of at most two molecules of cytochrorae a. Whether this represents a subunit of a larger complex, as it might be present in the mitochondrion, or whether there are three separate molecules of Complex [V per chain is a question which cannot be satisfactorily al~swered at present. The inhibitory effect of bilge acids on the enzymic activity of Complex IV has been shown by a number of autilors 19-°21. It was therefore not surprising that under the conditions ,xsed in these studies both enzymes exhibited very low activity. Biocl~im. Biophys. Aaa, 96 (1965) 1-8.

MOLECULAR V~EIGHTS OF COMPLEXES I I I AND I V TABLE tII EFFECT OF TAURODEOXYCHOLATE ON ACTIVITY OF COMPLEX I V

Conditions of assay

Velocity*

Per eem cx[ conlrol

Control Enzyme in~.u~ated in presence of 0.33 % taurodeoxych01ate fi~, 30 rain befo~ assay Enzyme incubated in presence ofo.33 % taurodeoxych~late for 30 rain a.,3d assayed in presence of 0.33 % t~.urodeoxyc]kolate

22.6

i oo

18.o

80

1-3

5.8

•/,molen of ferrocytocx~rome c oxidized per min per mg of protein. Table I I I ~-,howst h a t , when assayed in the presence of o.33 % taurodecxycholate, the a c t i v i t y of C,omplex IV was inhibited to the extent of 95%- However, when the enzyme was incubated with bile salt before assay, 80°o of tl~e original activity was restored. Furthermore, the a c t i v i t y was restored quickly and did not increase detectably d i n i n g the course of the assay reaction. Since aggreg~ tion was a relatively slow process it would appear t h a t dilution of the bile salt alone i.'. a sufficient condition of restoration of activity. Incubation of Complex IV in the presence of 0.33 % t a m o d e o x y c h o l a t c did not result in spectral change or in loss of reactivity t o w a r d c~rbo.a monoxide. The above evidence strongly supports, b u t does not establish the v~ew t h a t Complex IV, with a molecular weight of 23 ° ooo is a flmctional en;yme.

ACKNOWLEDGEMENTS We are indebted to Dr. D. E. Gr~EE:~ for his interest and criticism, to Dr, M. BUELL in the preparation of the manuscript, and to Mrs. P. KOCH for technical assistance. We t h a n k ttle Oscar Mayer Co. of Madison for their generous gifts of meat by-products. This investigation was supported in p a r t b y G r a d u a t e Tr~dning Grant 5-TI-GM88 and research grant GM-o5o73 from the National Instit~te of General Medical Sciences, U S P H S ; and b y Atomic Energy Comnfission Cont~ acts A T ( I I - I ) - 9 o 9 and A T ( I : t , I ) - I I 5 I. One of us (A. T.) was a post-doctoral trainee of the t n i v e r s i t y of Wisconsin I n s t i t u t e for E n z y m e Research; J. S. R. was a Researc:~ Career Dt~velopment Awardee, National Institutes of Health, U S P H (K-3-GM-2z.7;'4). REFERENCES

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8

A. TZAGOLOFF ~ a l ~

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B#ahim. Biophys. Aota, 96 (1t965) t-8