Vol. 80,No.

BIOCHEMICAL

3,1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 14,, 1978

Pages

CROSS-LINKING

553-559

STUDIES ON A CYTOCHROME -c -

CYTOCHROME c OXIDASE COMPLEX Margaret

M. Briggs

and Roderick

A. Capaldi

Institute

of Molecular Biology and Department of Biology University of Oregon Eugene, Oregon 97403

Received

December 27,

1977

SUMMARY A cytochrome c - cytochrome 2 oxidase complex containing 0.8-1.0 moles of cytochrome c per mole of cytochrome c oxidase (heme a + a ) was isolated as describe3 by Ferguson-Miller, S.,Brautigan, D.L., and3Margoliash E., J. Biol. Chem. 251, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 fi bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome c was cross-linked to subunit II of cytochrome c oxidase. Other cross-linked products were formed involving different subunits of cytochrome c oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. ExperTments are also described using N,N'-bis(3-syccinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 A bridging bifunctional reagent was a pair containing II+VI. INTRODUCTION Cytochrome atoms.

c oxidase

The enzyme catalyzes

and conserves of ATP [for

the

energy

review

cytochrome under

which

formed

several

of cytochrome

in this

redox

hemes and copper

c by molecular

reaction

between

and in which

cytochrome

c- - cytochrome

propionate

(DSP),

"active

a reagent

be used

Ferguson-Miller

complex

important

cleavable

for

oxygen

the synthesis

et al.

cytochrome

2 oxidase

[5]

reactive

553

complex

the binding

c is presumably

with groups

to

[4]. site

have described

5 and cytochrome

Here we describe complex

reagents

c oxidase

to determine

cytochrome

site".

with

bifunctional

in the cytochrome

can also

c on the enzyme. a tight

different

of subunits

approach

(Kd 10m7 M),

functionally

containing

see l-31.

the arrangement

The cross-linking

complex

the oxidation

liberated

We have been using study

is a multipeptide

conditions

c- oxidase

bound

for

is

at its

cross-linking

of this

dithiobissuccinimidyl 11 i apart

Copyright All rights

and bridged

by a

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

Vol. 80, No. 3, 1978

readily

cleavable

a newly

synthesized

BIOCHEMICAL

disulfide

tartaramide

in cytochrome

MATERIALS

Experiments

vie-glycol-bridged

oxycarbonylpropyl) units

bond.

AND BIOPHYSICAL

cross

are

RESEARCH COMMUNICATIONS

also

linker,

reported

in which

N,N'-bis(3-succinimidyl-

has been used to determine

near

neighbor

sub-

-c oxidase.

AND METHODS

Beef heart mitochondria were prepared by the method of Crane et al. [6]. Cytochrome 2 oxidase was isolated as described by Capaldi and Hayashi [7]. Preparations contained between 9.3-11.7 nmoles heme a/mg protein as determined by the pyridine hemochromagen difference spectral meThod of Williams [8]. Molecular activities were measured spectrophotometrically at 25°C as described by Vanneste et al. [9] and using an assay medium containing cytochrome c (25 PM), 0.5% Tween 80, 0.05 M potassium phosphate (pH 7.0). Values between 130-250 pmole cytochrome c oxidizedlseclumole heme ~3 were obtained for several different enzyme preparations. The cytochrome c - cytochrome c oxidase complex was prepared essentially as described by Ferguson-Miller et Tl. [5] but in a different buffer system. Cytochrome c oxidase (3-5 mg) was dialyzed against 25 mM HEPES, 0.25% Tween 20, 0.05% cliolate (pH 7.8) at 4°C to remove residual ammonium sulfate. A fivefold molar excess of cytochrome c (Type VI from Sigma) was added and the mixture (final volume 0.25 ml) was appligd to a column of Sephadex GlOO (18 x 1 cm) and eluted in the same buffer used in the dialysis step. The cytochrome c - cytochrome c oxidase complex eluted in the void volume , well separated from the excess cytochrome c. The concentration of each component in the complex was determined spectrally.The protein concentration was adjusted to 1 mg/ml and cross-linking was initiated by adding 0.1 to 1.0 mg/ml cross linker in (CH3)2SO. The reaction was allowed to proceed lo-30 min before quenching with 50 pl of a 1 M ammonium acetate solution. Also, 10 ~1 of a 10% solution of NEM was added to prevent disulfide exchange during analysis of the cross-linked products. SDS polyacrylamide gel electrophoresis in one and two dimensional systems was performed using the general procedures of Weber and Osborn [lo] and Swank and Munkres [ll] but with the modifications described before [4]. In some experiments a composite slab gel was used consisting of a lower layer of 20% acrylamide (plus 0.7% bisacrylamide) and an upper layer of 15% acrylamide (plus 0.5% bisacrylamide). Staining and destaining of gels was performed as described previously [4]. RESULTS A tight

complex

(Kd = 10e7 M),

per mole of cytochrome of Ferguson-Miller

aa

et al.

(2 hemes), [53.

2 oxidase

complex

was performed

different

incubation

times.

cross-linked

with

the Weber-Osborn

containing

was isolated

Cross

linking

with Figure

buffer

moles

cytochrome concentrations

1 shows the polypeptide for

conditions

10 min and then

but without

554

of cytochrome

by the general

of this

DSP at different

0.2 mg DSP/mg protein [lo]

0.8-1.0

profile

2

procedure c - cytochrome and for of a sample

run on a 12% gel

6 mercaptoethanol

present.

in

Vol. 80, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1 Two dimensional SDS polyacrylamide gel electrophoresis of the cytochrome c - cytochrome c oxidase complex to resolve the cross-linked with DSP. The upper gel trace shows the polyproducts formed by reaction peptide profile fo the cross-linked sample on a 12% gel in the Weber-Osborn buffer conditions but without B mercaptoethanol present. The slab gel shows the resolution of cross-linked products after they have been cleaved with B mercaptoethanol. Most of the untreated subunits of cytochrome c oxidase and any free cytochrome c runs on or close to a diagonal across the gel. Subunit III however runs off the diagonal (see Ref. 12). Also seen on the slab are several polypeptides present in small amount in cytochrome c oxidase preparations and these are labelled a, 6, and c. Our recent studies indicate that there are multiple copies of VII in the enzyme (Ludwig, B. and Capaldi, R.A., manuscript in preparation). The migration of VII on this two dimensional gel suggests that this band may contain different polypeptides of similar size. Components involved in cross-linked products are off and below the diagonal. Those which could be related to one another are circled. There are several off-diagonal spots for which a partner or partners could not be clearly resolved.

The major

bands

cytochrome

Cross-linked

c.

ran as shoulders Identification electrophoresing that

stained

seen were

unreacted products

to these

bands

polypeptides l),

were

or they

and analysis

in Figure

subunits

of cytochrome present

were

hidden

of cross-linked out

through

of an unstained a layer

555

but

products

of agarose

and free

in many instances

under

tube

coxidase

the

bands

themselves.

was achieved gel

(the

containing

they

by

duplicate f3 mercapto-

of

Vol. 80, No. 3, 1978

BIOCHEMICAL

ethanol,

and into

diagonal

in the slab

through

a slab

the agarose

monomer molecular

weights

faint

above or below

it.

concentrations

spot

of purified product

first

dimension,

just

which

range

of 37000-35000.

in this

in several

different

cross-linked

have been resolved

respectively) spot

weight

II.

under

of 33100.

identified

Other

clearly

of those

involving

study

before.

All

cytochrome

18 i between reactive of the (Smith,

of similar

[4].

c pair

cwere

must

be involved

Cross-linked

weights

Cytochrome

seen

in the

size.

36500

and 33400

2 was seen as a fairly has an aggregate

and I+V and IV+VII, listed,

molecular

IV+VI, which

included

which

just

2, were

seen in the control

spanning

groups.

ubiquinol

R.J.,

in preparation). cytochrome

weight

of the pairs

we have synthesized

DST and DSPT, the former

chain

high

with

was

have not been the exception (done

without

2 present).

Recently

studies

was

band in the

and these

molecular

also

directly

as cytochrome

range

all

products [4],

c.

by cross-linking

a molecular

II-cytochrome

subunit

cross-linked

in an earlier

resolved

cytochrome

This

1).

dimension

2 ran as a broad

(aggregate before

(Figure

DSP.

weight

products

of their

cytochrome

in the first

and V as well

molecular

II+V and III+V

involving

III

passed

as they

the diagonal

generated

I, with

II,

cleaved

contained

product

cytochrome

Subunits

to a

was seen on a vertical

5 with

of subunit

the diagonal

which

c dimer

containing

seen off

dark

of this

ran on or close

ran as a function

and below

no partner

cytochrome

ahead

then

resolved

of the cytochrome

The second

products

off

were

for

were

polypeptides

The migration

to that

products

to positions

products

One was a very

polypeptides

Cross-linked

and component

Two cross-linked

identical

Unreacted

gel.

gel.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

distance

These

R.A.,

c oxidase,

the cytochrome c containing gel

a maximal

have proved c reductase

Muchmore,

DST and DSPT were

c to cytochrome

two dimensional

vie-glycol

cytochrome

Capaldi, Both

two novel

in Figure

products

2, reaction 556

of 6 1 and the

segment

for

of the

DSP.

their

However,

in our

respiratory F.W.,

ability

generated

of cytochrome

latter

useful

D. and Dahlquist,

tested

did

cross-linkers,

particularly

but neither than

bridged

greater

manuscript

to cross-link amounts

as shown by the

c oxidase

with

DSPT

of

Vol.80,

BIOCHEMICAL

No.. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

IIf-HBE-

i I Lb+c

Figure 2 Two dimensional SDS polyacrylamide gel electrophoresis of cytochrome 2 oxidase cross-linked with 0.25 mg DSPT/mg enzyme for 30 min at room temperature. Cross-linked products were separated on a 12% gel in the Weher-Osborn gel conditions. The bifunctional reagent was cleaved by soaking the gel for 2 hr in several changes of a solution containing 0.015 M sodium periodate, 0.1% SDS 0.01 M sodium phosphate (pH 7.0). Electrophoresis in the second dimension was in the Swank-Munkres buffer system.

generated before

a cross-linked in studies

product

with

either

containing

the

II

and VI which

6 fi or 11 1 cross

has not been seen

linkers.

RESULTS The question cytochrome [133

of which

-c binding

and Bisson

-c and used this from

heart

this

cytochrome

[14]

mitochondria.

concluded

that between

site

According

c derivative in our

the binding

in several

have synthesized

to tag the binding

V and VII

of cytochrome

has been examined

et al.

(probably

linkage

subunits

for

site

for

cytochrome

lower

terminology).

[13]

studies.

Both

2 on cytochrome the

binding

site

subunits

In contrast,

Bisson

et al.

cwas

on subunit

in Erecinska

cytochrome

weight

cytochrome

557

involved

molecular

cytochrome

the photoaffinity-labelled

recent

are

a photoaffinity-labelled

to Erecinska

is on the

c oxidase

II.

2 and subunit(s)

c oxidase for

[14] The

8lOCHEMICAL

Vol. 80, No. 3, 1978

of cytochrome

c oxidase

the assignments changes

polypeptide

in the cross-linked

cytochrome

& oxidase

sulfhydral

group

[15]

bound

cross-linked

product

but

using

formed

and the

chrome

c oxidase

obtained

[133;

binding

[153).

results

then

subunit

II

oxidase

complex.

Several along

with

Figure

3.

lysine

It are

within

for

the

at its

from of components

a disulfide mild

with

beef

heart

in agreement

with

11 w of cytochrome

cross-linked

there

are

published

The cross-linking on the arrangement

the studies

of Eytan

data

[18J

in preparation),

558

III

enzyme [16])

is

were

identified

own experiments [35S]

on the

This

cross-

cyto-

is

product similar

cytochrome

with

c

II.

study.

summarized

the

other

c oxidase. (Ludwig,

Our

and place

- cytochrome

are with

cytochrome

[14]

in this

in

c (36000

subunit

et al.

results

in cytochrome

2

This

modified

shown are consistent

in which

subunit

The cross-linked

of Bisson

cross-linking

and our

by

by DSP.

of 37000-35000.

c in the cytochrome

of subunits

obtained

to cytochrome

several

c [17].

findings

products

to regenerate

c.

-c in association

the

cytochrome

The

c oxidase,

photoaffinity-labelled

cytochrome

bond.

The results

5,5'-dithiobis(2-nitrobenzoate)

contained

free

conditions

c was bound

of which

c to

This

two was stabilized

weight

et al.

modified

of the

cytochrome

molecular obtained

c,

cytochrome

of cytochrome

our previously

manuscript

of cytochrome

cytochrome

or III

face

and with

other

II

residues

information

R.A.,

inferred

the binding

under

in yeast

between

- binding

[14])

(38000

were

examination

identification.

that

site

interaction

with

direct

unmodified

to the product 36100

[13,14]

through

be cleaved

to subunit

had an apparent

in size

Therefore

conditions.

5,5'-dithiobis(2-nitrobenzoate).

could

indicate

experiments

reacts

mild

from direct

cytochrome

c oxidase

for

[15]

to the

linker

than

examined

yeast

to cytochrome

may be equivalent

oxidase

et al.

rather

with

polypeptides

et al.

In our

and Bisson

have also

by reaction

5 derivative

at or close

under

profile

et al.

the component

be cleaved

product.

Birchmeier

(which

not

made by Erecinska

in the

Birchmeier

could

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

c

These in available

For example B. and Capaldi,

DABS was used to label

c

BIOCHEMICAL

Vol. 80, No. 3, 1978

Figure 3 A schematic to date for beef heart

polypeptides

exposed

place

II,

III

these

components

of the membrane

drawing surrunarizing the cross-linking cytochrome c- oxidase.

at the different

and V exclusively are for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

close interaction

sides

on the

with

of the mitochondrial

outer

to one another

results

(intracristal)

and subunit

cytochrome

inner surface.

II

is

otained

membrane, Therefore

on the correct

side

-c in mitochondria.

ACKNOWLEDGEMENTS This research was supported by a National Science Foundation Grant PCM 74-00957, by NIH Grant HL 22050-01, and by a Grant in aid from the Oregon R.A.C. is an Established Investigator of the American Heart Association. Heart Association. REFERENCES 1. Caughey, W.S., Wallace, W.J., Volpe, J.A. and Yoshikawa, S. (1976) in The Enzymes (P-D. Boyer ed.) Academic Press, N.Y., 299-344. 2. Wikstrom,M.K.F., Harmon, H.J., Ingledew, W.J. and Chance, B. (1976) FEBS Lett. 6!& 259-277. Malmstrom, B.G., Quart. Rev. Biophys. (1973) 6, 389-429. :: Briggs, M.M. and Capaldi, R.A. (1977) BiochemTstry 16, 73-77. 5. Ferguson-Miller, S., Brautigan, D.L. and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115. 6. Crane, F.L., Glenn, J.L. and Green, D.E. (1956) Biochim. Biophys. Acta. 22, 475-487. Capaldi, R.A. and Hayashi, H. (1972) FEBS Lett. 2&, 261-264. ;: Williams, J.R. (1964) Arch. Biochem. Biophys. 107, 537-544. 9. Vanneste, W.H., Ysebaert-Vanneste, M. and Mason, H. (1974) J. Biol. Chem. 249, 7390-7401. 10. Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. Swank, R.T. and Munkres, K.D. (1971) Anal. Biochem. 39, 462-477. 11. 12. Capaldi, R.A., Bell, R.L. and Branchek, T. (1977) Bizhem. Biophys. Res. Commun. 74, 425-433. Erecinska, M. (1977) Biochem. Biphys. Res. Commun. 76, 495-501. It: Bisson, R., Gutweniger, H. Montecucco, C., Colonna, R., Zanotti, A. and Azzi, A. (1977) FEBS lett. g, 147-150. C.E. and Schatz, G. (1976) Proc. Nat. Acad. Sci. 15. Birchmeier, W., Kohler, USA 73, 4334-4338. 16. Brig?js, M.M. (1977) Ph.D. Thesis, University of Oregon. 17. Margoliash, E., Ferguson-Miller, S., Tulloss, J., Kang, C.H., Feinberg, D.L. and Morrison, M. (1973) Proc. Nat. Acad. Sci. USA B.A., Brautigan, 70, 3245-3249. G. and Racker, E. (1975) J. Biol. Chem. 18. Eytan, G.D., Carroll, R.C., Schatz, 250, 8598-8603. 559