Resolution and reconstitution of cytochrome oxidase

Vol. 90, No. 4, 1979 October

BIOCHEMICAL

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

Pages

29, 1979

RESOLUTION AND RECONSTITUTION

1119-1124

OF CYTOCHROME OXIDASE

Mitchell

Fry

Institute for Enzyme Research University of Wisconsin-Madison Madison, Wisconsin 53706 Received

August

27,

1979

SUMMARY

Cytochrome oxidase has been resolved into two fractions by extraction with formic acid. An insoluble-fraction (subunits I-III) retains the heme and phospholipid of the original enzyme and a soluble-fraction (subunits IV-VII) Recombination of the two enzymatically inactive fractions retains the copper. in the presence of sodium deoxycholate results in the expression of full enzymic activity. INTRODUCTION In previous distinct

fractions

separation (ITC)

subunits peptide

are

procedure associated fraction lipidated

apparently

(IV-VII)

amino

the

fraction

resolution

for

acids

smaller

fractions

were

procedure

also I-III)

into

appear

oxidase

mixture.

The

complex

to reflect

the physio-

largest

subunits

DNA whereas (3-6),

the

(I,

smaller

and the

two poly-

as reflected

by their

relative

Under

the conditions

of this

resolution

of the

(subunits

absent

I-III).

from

Both

completely

the

devoid

describes two fractions,

original

of the acid-soluble

the acid-insoluble acid-soluble,

and deacid-insoluble,

of enzymic for

comprising

in an aqueous

enzyme was found

IV-VII)

a new method

and a soluble-fraction

is completed

two

ion-transfer

- the

ribosomes

heme and copper

communication

subunits

(ETC),

polypeptides

(subunits

oxidase,

to as the

into

properties

(7,8).

completely

oxidase

an acid-alcohol

by mitochondrial

different

of the

and was almost

The present

(essentially

referred

on cytoplasmic

have very

of cytochrome

employing

in cytochrome coded

the majority with

resolution

complex

originate

of apolar

and recombined

cytochrome

two fractions,

inherent

classes

contents

the

electron-transfer

dichotomy

III)

(1,2)

has been described

of these

and the

logical II,

articles

activity. the resolution

of

an insoluble-fraction (subunits

environment

IV-VII).

This

in the absence

of

0006-291X/79/201119-06$01.00/0 1119

Copyright AI1 rights

@ 1979 by Academic Press, Inc. in onyform reserved.

of reproducrion

Vol. 90, No. 4, 1979

detergents activity

or dissociating from

the otherwise

BIOCHEMICAL

agents, inactive

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and can be reversed resolved

EXPERIMENTAL

to regain

full

enzymic

fractions.

PROCEDURES

Cytochrome oxidase was prepared by the method of Fowler et al. (9) as modified by Capaldi and Hayashi (10) and further purified accordzg to Fry et al. (1). Final preparations of cytochrome oxidase based on protein determina-on by the biuret method consistently had an average heme content of 12 nmol/ mg protein and 14 nmol Cu/mg protein. Estimation of copper was by the method of Felsenfeld (11) and heme estimation was as previously described (1). The method developed by Swank and Munkres (12) for sodium dodecyl sulfate/urea/gel electrophoresis in highly crosslinked gels was employed for analysis of the subunit composition of cytochrome oxidase. Gel electrophoresis procedures were identical to those recommended by Downer --. et al. (13) except that the staining time was doubled and protein samples were incubated in dissociating buffer for 1 hr. at 37°C. Routine protein estimation was by the method of Lowry --et al. (14) and phospholipid estimation by the method of Fleischer -et --al (15). Cytochrome oxidase activity was measured with a Clark oxygen electrode at 37'C. The assay medium (6 ml) contained 3.6 mg of cytochrome 2, 300 umoles of Tris-ascorbate (pH 6.8), 300 umoles of potassium phosphate buffer, pH 7.4, and was 3% (v/v) in Tween-80 detergent and 0.01% (v/v) in K+-cholate. Resolution of cytochrome oxidase. Prior to resolution, preparations of cytochrome oxldase were dialvzed aoainst 100 volumes of 50 mM ootasssium ohosohate The dialyzate'was centrifuged for buffer, pH 7.4, for at least 12 hrs at 4°C. 30 min at 30,000 rpm in a Spinco No. 30 rotor and the protein pellet was resuspended and washed by centrifugation (repeated three times) from 100 ml of distilled water. The protein pellet was resuspended each time by thorough homogenization. In a typical experiment, 50 mg of particulate cytochrome oxidase was sedimented in a 50 ml glass centrifuge tube and to the wet pellet was added 10 ml of 10% (v/v) formic acid, at 4'C. This mixture was vortexed for 20 set and allowed to stand on ice for 5 min. The contents were centrifuged in a refrigerated International centrifuge at 2,000 rpm for 10 min. The pale green supernatant (hereafter designated the "soluble-fraction") was removed with a Pasteur pipette. The pellet (hereafter designated the "insolublefraction") was resuspended in 5 ml of 10% formic acid, at 4'C, and centrifuged as before, the supernatant being combined with the first soluble-fraction. The insoluble-fraction was resuspended in 50 ml of 50 mM potassium phosphate buffer, pH 7.4, to which solid ammonium sulfate (to 25% saturation) was added and the whole again sedimented by centrifugation at 2,000 rpm for 15 min. This washing procedure was repeated once more without addition of ammonium sulfate. To the combined soluble-fraction (15 ml) was added 5 ml of a saturated solution of Tris-base plus 5 ml of a saturated ammonium sulfate solution (pH 7.8). This mixture was mixed and allowed to stand on ice for 5 min after which it was centrifuged at 2,000 rpm for 15 min. The pelleted solublefraction was washed twice as for the insoluble-fraction. Reconstitution of cytochrome oxidase activity. The soluble and insolublefractions were combined in 1 ml of 10% Na+-deoxvcholate. oH 8.5. Protein solubilization was aided by brief sonication (~5 min) in h bath-sonifier at room temperature followed by incubation at 37°C for 10 min. These procedures (sonication and incubation) were continued until complete solubilization was achieved. This solution was then diluted to 100 ml with distilled water and solid ammonium sulfate added to 25% saturation with continual stirring. The precipitated protein was sedimented by centrifugation at 30,000 rpm for 45 min

1120

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in a Spinco No. 30 rotor. Pelleted protein was resuspended in 100 ml 50 mM phosphate buffer, pH 7.4, and precipitated by addition of solid ammonium sulfate to 25% saturation as in the previous step. The final protein pellet was solubilized in a solution 50 mM in phosphate buffer, pH 7.4, 3% (v/v) in Tween80 and 0.01% (v/v) in K+-cholate, to a final protein concentration of about 10 mg/ml. Soluble and insoluble-fractions were also solubilized in the above solution for sampling of residual oxidase activity. Aside from treatment by formic acid, unresolved cytochrome oxidase was treated by the same reconstitution procedure in the presence of Nat-deoxycholate to solubilize the enzyme and for the purpose of direct comparison of enzymic activity with the reconstituted enzyme. RESULTS Densitometric fractions in

1.

of cytochrome

though

of the

along

smaller

whereas

polypeptides

of insolubility

(Fig.

present

of the cytochrome removal

of cholate

2).

was found

tion

to an insoluble-fraction

copper with

insoluble-fraction

as traces

of other

of subunit

prior

the with

small

sub-

on the degree

to resolution. oxidase

a greater

con-

IV and of

depends

cytochrome

containing

smaller in both

to correlate

preparation

sub-

distributed

of the

original

shown

the

The small

The amount

the

are

contains

1) appears

oxidase

from

resolution

soluble-fraction.

(as well

and soluble-

of the larger

in the insoluble-fraction

Incomplete leads

oxidase

subunit

the copper

acid

essentially

and re-extraction

this

insoluble

soluble-fraction

(see Table

IV present

with

the

in the

the

by formic

IV of cytochrome

removes

for

consists

insoluble-fraction

acid

profiles

obtained

predominantly

of subunit

10% formic units)

oxidase

Subunit

fractions

amount

oxidase

The insoluble-fraction

polypeptides.

tent

of the gel

of cytochrome

Fig.

units

traces

extent

prepara-

of the

smaller

polypeptides. Compositions (similar

to those

and respiratory

shorun in Fig. 1) are

phospholipid

are

concentrated

in the

tions

are clearly

Neither constitution

fraction

activity

concentrated

soluble-fraction.

rationalized possessed

regained

in the

of the

summarized

reconstituted in Table

insoluble-fraction The protein

on the significant

basis

respiratory

some 93% of the original

1121

1.

Heme -a and

whereas

copper

contents

of their

subunit activity

activity.

fractions

was

of the two fraccompositions. alone

but

If cytochrome

upon reoxi-

BIOCHEMICAL

Vol. 90, No. 4, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

L A

Pd

Cu content (nmdes.mg.prot.1 44

C

L---

01

-.

02

+

-Migration

Densitometric by formic acid w enved B. Insoluble-fraction.

traces of gel profiles of extraction. A. Unresolved C. Soluble-fraction .

NMigration

+

oxidase and

cytochrome cytochrome

fractions

oxidase.

Densitometric traces of gel profiles of reextracted insoluble-fraction Fig. 2. of cytochrome oxidase. A. Insoluble-fraction resolved by one formic acid extraction. B. Insoluble-fraction reextracted once in formic acid. C. Insoluble-fraction reextracted twice by formic acid.

I

TABLE STRUCTURAL

AND FUNCTIONAL

Parameter

Total

protein

PARAMETERS

OF RESOLVED

AND UNRESOLVED

CYTOCHROME

OXIDASE

Unresolved enzyme

Solublefraction

Insolublefraction

50

21.5

28.3

49.8

19.1

11.7

(mg)

Heme a (nmoles/mg pr0t.T

12.2

3.1

Reconstituted enzyme

13.9

Cu (nmoles/mg

prot.)

14.5

27.7

4.4

Phospholipid (i-&4. wet.

content 1

59.1

5.8

99.6

59.1

15.8

CO.5

<1.2

14.7

<3

C7.5

93

Respiratory activity (uatoms O/min/mg. Respiratory (% recovery

dase

prot.)

activity of original)

was

subjected

100

to

10%

base and ammonium sulfate attempt

to

separate

the

formic

and

acid

(as done for two

fractions,

immediately

the

soluble-fraction),

then

reconstituted

1122

neutralized

with

Tris-

and without activities

any

of 95-

BIOCHEMICAL

Vol. 90, No. 4, 1979

100% of the original

could

with

Na+-deoxycholate).

tion

with

led

formic

to lesser

and precipitation

resolution two layers,

an upper

green

(the

layer

with

with

of these

centrifugation

green

layer

that

acid

followed

and the

of the

by immediate

the

result

pellet

soluble-fraction)

appearance

original

of the

consists

of

and a lower

of success

can be judged

Nat-deoxycholate

from

the soluble-fractions,

the protein

(the

insoluble-frac-

(65-85%).

The degree

two layers

indistinguishable

formic

treatment

the

one to visualize

insoluble-fraction).

by solubilization appearance

pale

with

activity

allows

Following

method.

reconstitution

by recombination

oxidase

RESEARCH COMMUNICATIONS

to "clean-up"

in reconstituted

of cytochrome

neutralization

(following

extraction

followed

recoveries

Treatment

be regained Further

acid,

AND BIOPHYSICAL

of reconstitution

by the

subsequent

of an homogeneous

cytochrome

dark

dis-

fraction

oxidase.

DISCUSSION Past lytic

activity

lipidation free

at resolution

enzyme complex

heme and copper

(see

these

subunits

were

The present

certainties

by the almost

of the

enzyme complex

solubilization Locali'zatSon

complete

agreement

used conditions

is

originally

a method

gentle

and fully

of heme with the

designed

recent

larger findings

to mitigate

of phospholipid

with

in standing

with

nature

the

larger

of these

1123

with

of cata-

to dissociation

and de-

and reattachment

therein).

Subsequent

therefore

rested

associated

with

both

simple

reversible

subunits

to avoid

these

un-

in design.

activity,

and leads

resolution to the selec-

polypeptides. of cytochrome

of Freedman against

identi-

prosthetic

and quick

of enzymic

of

on the assumption those

has been devised is

retention

release

and more polar

the

association

the apolar

which

reconstitution

of the smaller

with

consequent

technique

complete

can lead

subunits

indeed

and to provide

oxidase

16 and references

resolution

Judged

also

ref.

that

with

of heme or copper-binding

groups.

tive

of cytochrome

have been made in media of the

fication that

attempts

--et al.

migration

subunits polypeptides.

oxidase (17)

of free

of cytochrome

is

in

who have heme. oxidase

The is

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

results

support

idea

of a hydrophobic

"core"

The present consists which

are arranged

heme moiety

smaller

presumably

undefined

lies

association.

of cytochrome peptides

oxidase

which

seems likely membrane chrome

the

phase oxidase

tent

with

our

form

an active

on or within

to enclose

findings

This

of small

polypeptides

those

picture

entities

of the enzyme

when incorporated

nature across

the

by an as yet largest

an assembly

III

around

The hydrophobic

that

is

complex

phospholipid,

structure

(18)

subunit

on the transmembraneous

ion-channel

central

shown

with

with

oxidase

polypeptides.

I, % M Wt. 40,000)

and retain

complex.

this

recently

be identified assembly

the cytochrome

association

and more polar

(subunit

this

that

in tight

We have

may also that

the

RESEARCH COMMUNICATIONS

subunit

of smaller

It

(QM Wt. 20,000). is

that

arranged form

structure of subunit liposomal

within

the

the active is

also

cytoconsis-

I that

can

membranes

(2).

ACKNOWLEDGEMENTS The author E. Green.

This

GM12847 of the

gratefully

acknowledges

investigation National

Institute

the

was supported of General

support

and interest

in part

by Program

Medical

Sciences.

of Dr. Project

David Grant

REFERENCES 1. 2. i: 5. 6. 7. 8. 9. 1:: 1:: 14. 15. 16. 17. 18.

Proc. Natl. Acad. Sci. Fry, M., Vande Zande, H. and Green, D. E. (1978). USA 75, 5908-5911. Fry, M. and Green, D. E. (1979). Proc. Natl. Acad. Sc4 .USA 76, 2664-2668. Mahler, H. R. (1973). CRC Crit. Rev. Biochem. 1, 381-460. Schatz, G. and Mason, T. L. (1974).Ann. Rev. Biochem. 43, 51-87. Rubin, M. S. and Tzagoloff, A. (1973). J. Biol. Chem. 248, 4269-4279 _ Sebald, W , Machleidt, W. and Otto, J. (1973). Eur. J. Biochem. 38, 311324. Poyton, R. 0. and Schatz, G. (1975). J. Biol. Chem. 250, 752-761. Kraml, J. and Mahler, H. R. (1967). Immunochemistry 4, 213-226. Fowler, L. R., Richardson, S. H. and Hatefi, Y. (1962). Biochim. Biophys. Acta 64, 170-173. Capaldi, R. A. and Hayashi, H. (1972) FEBS Lett. 26, 261-263. Arch. Biochem. Biophys. 87, 247-251. Felsenfeld, G. (1960). Swank, R. J. and Munkres, K. D. (1971). Anal. Biochem. 39, 462-477. Downer, N. W., Robinson, N. C. and Capaldi, R. A. (1976). Biochemistry 15, 2930-2936. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Fleischer, S., Brierley, G., Klouwen, H. and Slautterback, D. B. (1962). J. Biol. Chem. 237, 3264-3272. Phan, S. H. and Mahler, H. R. (1976). J. Biol. Chem. 251, 270-276. J. Biol. Chem. 254, Freedman, J. A., Tracy, R. P. and Chan, S.H.P. (1979). 4305-4308. G. A. and Green, D. E. (1979). FEBS Lett. in press. Fry, M., Blondin,

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