Purification of cytochrome b from Complex III of beef heart mitochondria

Vol.

132,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

November15.1985

PURIFICATION

OF CYTOCHROME b FROM COMPLEX III

Hideto

Nakahara,

Department

Received

1166-1173

Pages

Yoshiharu

Shimomura,

of Biomedical University of

September

23,

OF BEEF HEART MITOCHONDRIA and

Takayuki

Chemistry. Faculty Nagoya, Nagoya. 466

Ozawa

of

Medicine,

Japan

1985

Two cytochrome b preparations have been prepared from Complex III of Summary: beef heart mitochondria, by detergent-exchange chromatography on a butylToyopearl column. One was eluted from the column with buffer containing Tween 20 after most of other subunits of Complex III were eluted with buffer containing guanidine-HCl, and the other was eluted from the column with buffer The former is consisted of a single polycontaining sodium dodecyl sulfate. peptide (subunit III) and contained 37.5 nmol of heme b/mg of protein, and the latter consisted of subunits III and IX and contained 19.5 nmol of heme b/mg The former was labile when it was reduced by dithionite, whereas of protein. the latter was stable. Subunit IX in the latter is associated with cytochrome b even after gel filtration and density gradient centrifugation. These results suggest that subunit IX plays a role in stabilizing cytochrome b. @ 1985 Academic Press,

Inc.

Cytochrome

b

(cytochrome

bcl

cytochrome

c

two

of

types

presumed

has

the

i-+

and

energy

of

in

bT,

the

by

the

Although

cytochrome

studied

which

ubiquinol

the

b and

many

other

to

presence

of

chain

was

electron-transfer (2).

between

from

from

III

Complex

transport

behavior

transduction

intensively

mitochondrial

electron redox

interaction

been

components

catalyzes

b, to

have

the

peculiar

cytochrome

III

remain

function

of

subunits

in

investigators

(3),

they

obscure. Our

III

by

sulfur by

and

b and

of

which

relation

cytochrome

one

complex) (1).

in

Complex

is

group

has

been

disassembling protein

and

(4)

and

study,

Complex

III

we using

column.

polypeptide

and

Copyright AN rights

cl

addressed

the

structure

and

the

complex.

Isolation

from on

ourselves

a less Here

the

(5)

chromatography

Sepharose

0006-291X/85

reassembling

cytochrome

detergent-exchange

present

investigating

we

report

cytochrome

interaction that

associated

$1.50 0 1985 by Academic Press, of reproduction m any form

Inc. reserved.

isolation

1166

cytochrome with

was

so

Complex the

far

column

In

cytochrome

b

than

b composed

IX of

ironachieved

column. of

subunit

of of

a phenyl-Sepharose to

hydrophobic

III

Complex

function

the of Complex

the from

phenyla

single

III

are

Vol.

132,No.

obtained

by

that

detergent-exchange

the

chrome

BlOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

3, 1985

latter

b was

chromatography

preparation

is

more

on

stable

than

and

Methods

a butyl-Toyopearl the

former

column, one,

when

and cyto-

reduced. Materials

Materials Complex III was prepared from beef heart mitochondria by the method of Rieske et al. (6). A bc, subcomplex (devoid of the iron-sulfur and subunit X), which contained 7.4 nmol of cytochrome b/mg of protein was prepared from Complex III according to the method of Shimomura protein, Chemicals, Ltd., and Ozawa (7). Guanidine-HCl was purchased from Nakarai Kyoto; Tween 20 from Wako Pure Chemical Industries, Ltd., Osaka: cholate from Sigma, St. Louis. Cholate was recrystallized from 50% hot ethanol before use. Butyl-Toyopearl 650M was obtained from Toyo Soda MFG. Co., Ltd., Tokyo. Sephacryl S-300 superfine was the product of Pharmacia Fine Chemicals, Other chemicals used were of reasent arade. Uppsala. Purification of cytochrome b All the foliowing procedures were carried Buffer A used in the followina out at O-4"& unless otherwise stated. experiment was composed of 50 mM Tris-HCl, pH 8.0, 0.5% cholate, 20% glycerol: The bc, subcomplex (50 mg of protein) in 25 mM and 1 mM dithiothreitol. pH 7.5, containing 0.5% cholate, 3.2 M NaCl, and 20% glycerol was Tris-HCl, applied to a butyl-Toyopearl column (1.5 x 6 cm) equilibrated with Buffer A containing 3 M NaCl. After washing the column with 50 ml of the same buffer, 20 ml of Buffer A and 20 ml of 2.7 M guanidine-HCl dissolved in Buffer A was successively passed through the column, resulting in elution of pink-colored fractions (mainly cytochrome c core proteins, and subunit VI). After the guanidine-HCl was washed out w-!ih 12 ml of Buffer A, a brownish-pink-colored fraction (cytochrome t$ was eluted with 40 ml of Buffer A containing 3% Tween Tween 20 on the column was removed by washing the column with 25 ml of 20. Buffer A, and subsequently pink-colored fraction (mainly cytochrome and El subunit VIII) was again eluted with 17 ml of 3.4 M guanidine-HCl dissolved in Buffer A. Guanidine-HCl was washed out using 14 ml of Buffer A, and the remaining protein (mainly cytochrome b and subunit IX) on the column was finally eluted at room temperature with Buffer A containing 2% SDS'. Cytochrome b was Analytical Methods estimated from the difference of the dithionite-reduced minus absorption spectrum ferricyanide-oxidized Absorbance measurements were performed using a Shimadzu UV-VIS form. recording spectrophotometer, model UV-250. The millimolar extinction coefficient used was 28.5 for A (8). The heme content was also determined by 562-57{ the pyridine hemochromogen spec rum using a millimolar extinction coefficient in the reduced form (9). Protein was measured by Lowry's ;:th:: f::d:?;$d60g y Hartree using bovine serum albumin as a standard (10). Polyacrylamide gel electrophoresis in the presence of SDS, urea, and glycerol was performed as described by Merle and Kadenbach (11). except that samples were treated in 10 mM Tris-phosphate buffer, pH 8.3, containing 4% SDS, B M urea, 10% glycerol, and 40 mM dithiothreitol at 37°C for 1 h. After electrophoresis the gel was stained with Coomassie Brilliant Blue. Results Since and

subunit

subcomplex

1

The

Complex

III

X when

most

(devoid

abbreviation

easily of

and

Discussion

dissociates

the

phospholipids

of

these

two

subunits)

used

is:

SDS,

sodium

of

the

was

dodecyl 1167

iron-sulfur complex used

as

sulfate.

protein are a starting

removed

(subunit (7). material

V) a bcl for

Vol.

132,

No.

BIOCHEMICAL

3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A

01

o 1

IO

20

30

Fraction

40

50

B

C

D

02

60

Number

Fig. 1. Detergent-exchange chromatography of a bc subcomplex. After binding the bc, subcomplex to a butyl-Toyopearl 650M -1 co umn. cytochrome cl, core proteins. subunit VI were eluted with Buffer A (see "Materials and Methods") (arrow a) and 2.7 M guanidine-HCl in Buffer A (arrow b). Guanidine-HCl was washed out from the column with Buffer A (arrow c). and a cytochrome b fraction was then eluted with Buffer A containing 3% Tween 20 (arrow d). After the column was re-equilibrated with Buffer A (arrow e), residual cytochrome cz, and subunit VIII was eluted with 3.4 M guanidine-HCl in Buffer A (arrow f). Guanidine-HCl was washed out from the column with Buffer A (arrow g), and finally the remaining proteins (mainly cytochrome b and subunit IX) were eluted with Buffer A containing 2% SDS (arrow h). -, absorbance at 280 nm; at 416 nm. Absorbance at 280 nm of the cytochrome ~QI fraction eluted ----1 with Buffer A containing 3% Tween 20 is the sum of absorbance of protein and - 0 14/%/cm). Approximately 5 ml of fractions were collected at k?rfk?e(~?8!!O-mlih. F ractions shown by the horizontal bar were recovered.

III. ug);

Fiq. 2. SDS-polyacrylamide gel electrophoretic patterns of Complex and two cytochrome b preparations, subcomplex, A, Complex III (13 subcomplex (9 ug); C, the cytochrome b preparation eluted with bc, (2 ug); D. the cytochrome b preparation eluted with SDS (3 ug).

purification

of

to

detergent-exchange

The

bc,

subcomplex

protein

3

was When 2.7

column,

and

in

this

was in

bound NaCl

to was

the

mainly

cytochrome

subunit

VI,

were

on

III

out

subcomplex

from

succinate

Buffer

Buffer

A and

(subunit

IV),

(electrophoretic

1168

A b)

second

peaks

core

2A). A

a

was

in

1).

in

the

containing 1)

and the

through Fig.

(subunits not

of

Fig.

passed

proteins data

All

Buffer

(arrow

(arrow

(Fig. dehydrogenase

(Fig. with

the 20

subjected

column

preparation

using

was

butyl-Toyopearl

equilibrated

(first

eluted

bc,

free

column

in

IZ,

a

completely

washed

fractions

The

study.

Complex

M guanidine-HCl

pink-colored

contained

2B)

contaminated

NaCl.

subsequently

II),

(Fig.

applied M

b

chromatography

70,000)

Or

cytochrome

&I

B, Tween

shown).

1).

which I

and After

Vol.

132,

guanidine-HCl with

was

Buffer

(arrow

the

washed

out

finally

eluted

the III

possibility tion

is

a

cytochrome

components

2B).

and

tion

was

cannot

be

ruled

This

the

view

with

of Et-

74,000

in 3A

a single

the

of

and

531,

form

has (A4l

the

The

562

occur nm.

maxima

at in

of

the the

also

at

of

difference

531

and

oxidized cytochrome

the

562

not

the

bcl

subcomplex

the

cytochrome

and as

(Fig.

416

nm,

and

spectrum nm. form)

of

This of

subunit

IX, VIII.

a dimer

of

those the

of

1.7.

This purified

(Fig. preparathose may of

and

b.

composed

oxidized

ratio

an is

by

form maxima form

minus

gives

minor

cytochrome

reduced

reduced

A

band

absorption the

a

The

b

the

be

Buffer

cytochrome

preparation

b preparations 1169

with

The

of to

composed

subunit

4).

dimer

it

eluted

in

the

than

b

is

by

prepara-

b

findings,

purified

two

correspond

urea

and

purified

thus

and

The

does

b and c,,

dithionite

279

band

considered

cytochrome by

a

cytochrome

cytochrome

spectra

reduced

6'A279

(1.28-1.38)

is

be

finally

cytochrome

absorption

form and

74,000,

might

these

only

b fraction

mainly

fraction

immediately

oxidized

428,

this

shows

of

it

SDS

From

contained

cytochrome

&-

polypeptide.

be

Methods."

preparation

with

of

the

Although the

when

concentrations

was

cytochrome

in

that

increaed

largely

74,000

material,

band

IX

of

74,000.

Mr

this

was

obtained'

of

&-

with

that

starting

this

lower

The

a band

Fig.

can

the

by

residual

subunit

patterns

with

suggests

supported

2% SDS consisted

amounts

Mr

with

h).

composed

band

out

guanidine-HCl

b preparation

was

washed

of

b and

(arrow

band

the

this

"Materials this

a minor

was

eluted

treated

After

cytochrome 20

20

elation

electrophoretic

The

that

in

shown).

was

further

cytochrome

Tween

out

in

with

polypeptide.

containing

b-

Tween

were

2% SDS

COMMUNICATIONS

b fraction

resulting

not

gel

and

is

amount

that

f),

RESEARCH

After

column

containing

contaminant,

under

single

those

37,000)

d).

the

(data

containing

Mr

treated

concluded

(arrow

A (arrow

cytochrome

observed

described

a cytochrome

A containing

with

that

c),

SDS-polyacrylamide

true

any

BIOPHYSICAL

on

VIII

Buffer

buffer

b.

20

a fraction

containing

b (subunit

ratio

g),

with

with

Buffer

subunit

2 shows

fractions

of

and

AND

(arrow

remaining in

(arrow

Fig.

and

proteins

Cl

elution

out

3% Tween

M guanidine-HCl

cytochrome

of

washed

A containing

e),

3.4

BIOCHEMICAL

No. 3, 1985

oxidized absorbance

higher other

at

than investi-

Vol.

132,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICALRESEARCH

Wovetength

COMMUNICATIONS

(nm)

3. Absorption spectra of the purified cytochrome b preparation and the cytochrome b preparation containing subunit IX. A, the purified cytochrome b preparation. The spectra were taken on the preparation (0.11 mg/ml) immediately after the elution from the column. The spectrum of the reduced form (370-650 nm) was separately recorded between 370-450 nm and between 450-650 the cytochrome b in the reduced form was very labile as described in nm. since the text. B, the cytochrome b preparation containing subunit IX. The spectra were taken on the preparation diluted g-fold with Buffer A (0.16 mg/ml). -, the oxidized form: ----, the dithionite-reduced form. The insets show each difference spectrum of the reduced minus oxidized form. Fig.

gators of

(12). protein

when

obtained ing

by that

of

containing those

of

the

between the affected

cytochrome

of

purified

protein by

from

the

pyridine

the

The

of

specific from two

values

in

this

treatment

the

when

subcomplex

pyridine may

maxima

31

be due

carbon

of

form)

nmol/mg

to

the

monoxide 1170

well.

not

b are

of The

preparation identical

the yield

to gives

contained

an 19.5

spectrum.

was This

hemes

the

shown).

difference

protein

of

of

20%.

and

the

was

suggest-

preparation

1.3

of

value

spectrum

(data

spectrum.

spectrum as

The

This of

nmol/mg

spectrum,

spectra

contamination The

same

cytochrome

from

hemochromogen

preparation. of

the

37.5

approximately

above.

oxidized

of

was

of

estimated

content

The

monoxide

carbon

b described

protein

the

spectrum.

of &T

was

hemochromogen

spectra

in

preparation

heme.

absorption

heme

this

no dissociated

treatment

absorption

(A416/A279

estimation these

difference

cytochrome

heme/mg the

the

by

IX.

of

from

b from

shows

content

contains

affected

ratio

However, the

was

subunit

absorbance nmol

estimation

38

Fig.

heme

preparation

form

yield

specific estimated

the

this

reduced The

The

obtained discrepancy

dissociated reduced of

by

from form

cytochrome

was b

BIOCHEMICAL

Vol. 132. No. 3, 1985

in

this

of

the

preparation

from

specific

heme

Although are

similar,

not

so

former

stable heme

purified

but the

not

purified

re-addition

of

dithionite.

absorption

bands

of

cytochrome

b preparation

slower has tion.

than no

of

the

This

effect

on

the

Since

cytochrome

were

the

Fig.

other

subunit

after

of has

the

was

significantly

increased

by

a and

after

cytochrome to

be

Fiq. 4. Absorbance changes in Soret tions after reduction with dithionite. cytochrome b preparation containing trations showing the same absorbance spectra to the right of the traces approximately 3 min after reduction.

was

of

Tween

b

preparaa

single

428

bands of the two cytochrome b preparaThe purified cytochrome b (I) and the subunit IX (II) were used a? the concenat 416 nm in the oxidized forms. The show the peaks of the absorption bands

1171

the

reduction

dithionite

Addition

B

of

NozSzO4 I

428 with

absorbance

of

reported

and at

on the

stable

I).

the

reduction

not

the

addition

IX:

absorbance

observed

stable

preparations

freezing

after

very

been

an

was

b (curve

III

after

4,

hand,

IX was

rate

Complex

was

also

also

basis

polypeptide

spectrum

in

the

subunit

decreased

phenomena On

b

a single

absorbance

properties in

of

on

spectrum.

cytochrome

and

shown

cytochrome

spectral

two

30%

containing

decreased

reduction

b

the

immediately

cytochrome.

purified

difference

difference

As

b was

the

the

temperature

one.

containing

although that

the

room

These the

approximately

b preparation

latter

I).

was

h composed

from

cytochrome

(curve

II),

of

at

the

dithionite

(curve

properties

cytochrome

reduction

was

from

estimated

after

thawing,

estimated

cytochrome

the

contents

diminished

nm of

spectral

as

subcomplex

bc,

content

the the

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20

Vol.

132,

No. 3, 1985

polypeptide the

which

two

the

stabilization

to

the

stabilization,

this,

the

IX.

buffer,

and

sucrose

in

8.0,

containing

was

performed

and

subunit Mr

same

not

shown),

these

at

cytochrome

be

S-300

gradient

density gradient

fraction

in at

indicating it

It

b. b and

cytochrome

the

If

latter

suggested

is

an

11, cytochromes

subunit the

20,

0.1

elution

one,

the

IX was

that

is

(1.5

and

Tween

20

was

used

and

subunits

III

would

be

concluded

bT,

pH

M

in

sucrose

role

the

interaction

related

with

(data ifi

in

redox

b

obtained

cytochrome

a

from

concentra-

(corresponding

also

0.4 with

as

centrifugation

ml

were

associated

glycerol

cytochrome

102

approximately

whether

Complex and

of

0.1

mM Tris-HCl,

analysis,

volume

gel

using

the

50

former

examine to

cm)

which

M in

subject

contribute

20%

0.7

IX plays

responsible

x 116

M NaCl,

subunit

be

subjected

to

of

preparation

To

0.2

two

b

would

in

In the

h.

between

cytochrome.

column

0.2%

IX

bK

may

subunit

from

interesting

IX in

small with

the

subunit

subunit

centrifugation

concentration

is

the

difference

cytochrome

small

Tween

48

at

that

subunit

b.

and x g for

the

this

ranged

co-eluted

and

of

COMMUNICATIONS

the

stable

superfine

to

110,000

the

containing

0.2%

the

that

(13-16).

associated

containing

IX were

facts,

cytochrome

must

1% deoxycholate,

250,000);

the

cytochrome

BIOPHYSICALRESEARCH

centers

is

a Sephacryl

pH 8.0,

of

b heme

b preparation

on

tion

AND

presence

it

chromatography

elution

The of

cytochrome

M Tris-HCl,

two

b preparations

subunit

for

of

contains

cytochrome

contains

to

BIOCHEMICAL

From

stabilizing between the

speed

two of

types b

heme

(17). References 1. z:

4. 5. 6.

Rieske, J.S. (1976) Biochim. von Jagow, G, and Sebald, Papa, S. (1976) Biochim. Shimomura, Y., Nishikimi, 14059-14063. Shimomura, Y., Nishikimi, press. Rieske, J.S., Zaugg, W.S.,

Biophys. Acta 456, 195-247. W., (1980) Ann. Rev. Biochem. 3, Biophys. Acta 456, 39-84. T. (1984) J. Biol. M., and Ozawa, M., and

and

Ozawa,

T. R.E.

Hansen,

281-314. Chem.

259,

(1985)

J.

Biol.

Chem.

in

(1964)

J. Biol.

Chem.

239,

3023-3030. 7. 8. 9.

Shimomura, Y., and Ozawa, Zaugg, W.S., and Rieske, 213-217. Theorell, H., Paul, K.G.,

T.

(1982)

J.S.

(1962)

and

Akeson,

Biochem. Biochem. A.

(1953)

Int. 5, Biophys. Acta

l-6. Res. Chem.

Commun. Stand.

1284-1287. 10. 11. 12.

Hartree, Merle, Weiss,

P., H.

E.F. (1972) Anal. and Kadenbach, (1976) Biochim.

Biochem. B. (1980) Riophys. 1172

4&, Eur. Acta

422-427. J. Biochem. 456, 291-313.

105,

499-507.

2, 1.

Vol.

13. 14.

15. 16. 17.

132,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

von Jagow, G., Sch'a'gger, H., Engle, W.D., and Machleidt, I. (1978) FEBS Lett. 91, 121-125. Slater, E.C. (1981) in Chemiosmotic Proton Circuits in Biological Membranes (Skulachev, V.P., and Hinkle, P.C., eds.), pp. 69-104, Addison-Wesley Publishing Company, Inc., London. T'sai, A.L., and Palmer, G. (1982) Biochim. Biophys. Acta 681, 484-495. Widget-, W.R., Cramer, W.A., Herrmann, R.G., and Trebst, A. (1984) Proc. Natl. Acad. Sci. USA 81, 674-678. Chance, B., Wilson, D.F., Outton, P.L., and Ereciiska, M. (1970) Proc. Natl. Acad. Sci. USA 3, 11751182.

1173