Isolation of QP-C and reconstitution of the QH2-c reductase

Vol. 104, No. 2, 1982 January 29, 1982

BIOCHEMICAL

ISOLATIOX

OF QP-C AN! RECONSTITUTION

Department State University

Received

December

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 591-596

OF THE QHz-c REDUCTASE

By Tsinp-ying Wang* and Tsoo E. King of Chemistry and Laboratory of Bioenergetics of New York at Albany, Albany, New York

12222

4, 1981

Summary A ubiquinone protein, QP-C, which acts in the cytochrome b-c .____ region has been solubilized. The isolated QP-C shows one band of molecules weight 15,000 in polyacrylamide gel electrophoresis in sodium dodecyl sulfate and isoelectric focusing at the isoelectric point of pH 3.6. QP-C reconstitutes with the QP-C deficient complex to restore the OH2 -cytochrome c --b-c activity and recover the EPR sign& of the complex.

It

has been

shown

Evidence

from

bound'. tory

chain

the chain

contaics (l-3).

dehydrogenase

many lines

at least

(SDH) to form radical

we have worked

out

tein,

acting

or OP-C,

in

that

isolated

at

reductase

systems

has been

(4).

the cytochrome

--b-c l region.

protein respiraloci

with

another This

of QH2-z

(5).

of the Recently,

ubiquinone paper

on

succinate

The formation

demonstrated

and purify

reconstitution

are

the different

and can reconstitute

to solubilize

of @P-C and subsequent

, (Q)l

the mitochondrial

such OP acting

succinate-Q

in these

a method

indicates

three

OP-S has been

ubisemiquinone

isolation

that

INTRODUCTION the active forms of ubiquinone

reports

prothe

reductase.

MATERIALS AND METHODS The cytochrome complex or QH2-cytochrome c reductase was pre--b-cl-111 pared from the --b-cl-II complex (2), and its activity was determined by a previously published method (6). The OP-C deficient b-c -III complex or the b-c -IV complex was prepared from digestion of the --b-cl: 4 II complex with imthat approximately 25-30% of the zoz&ized chymotrypsin at 30" to the extent *Exchange scholar with The People's Republic of China, permanent address: Shanghai Institute of Biochemistry, Academia Sinica, Shanghai. 1 Abbreviations used: DCIP, 2, 4, dichlorophenolindophenol; HPLC, high perthe subscript of Q denotes formance liquid chromatography; 0, ubiquinone; QH2* completely reduced 0; QP, ubithe number of isoprene units contained; quinone protein; OP-C, the QP functions in the region of the cytochrome complex; QP-N, the QP in the NADH dehydrogenase respiratory segment: --b-c sulfate; 0P-8, the immediate electron acceptor of SDH; SDS, sodium dodecyl and SDH, reconstitutively active succinate dehydrogenase.

591

0006-291U82/020591-06$01.00/o Copyright 0 1982 bv Academic Pres, Inc. All rrghrs ofreproducrron m any form reserved.

Vol. 104, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

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original activity remained. Usually 45-60 minutes were required, incubating mixture of immobilized chymotrypsin and the --b-cl -III complex in a ratio of 1 g wet weight of immobilized chymotrypsin which was obtained by filtration on a medium porosity sintered glass funnel per 4 ml of the b-cl-III complex conThe mixture was filtered to rem&e the proteolytic taining 6.6 mg. per ml. enzyme. The immobilized chymotrypsin was prepared by reacting 20 mg of three times recrystallized enzyme per g CNBr-activated sepharose 4B (Pharmacia) in 0.1 M bicarbonate buffer pH 8.0 for 2 hrs at 22-24". The excess of activating group was destroyed by addition of 0.2 M glycine buffer pH 8.0 according to the manufacturer's manual. 02 was synthesized in this laboratory by a modification of the method of Shunk, -et -*al Q2H2 was pre(7). pared according to Engel, --et al. (6). OP-C activity was assayed by incubating the isolated QP-C rich fractions with the --b-cl-IV complex. The activity of QH,,-2 reductase was determined with aliquots drawn from the incubation mixtu?e by measuring the reduction of cytochrome c at room temperature. See the Results and Discussion section for further details. All other materials were procured from the highest purity commercially available. The water used was double distilled in an all glass apparatus and deionized. The EPR measurements The machine setting legend of Fig. 2 in ref.

were made with Varian E4 as previously described was made at room temperature as described in the (8). RESULTS AND DISCUSSION

(8).

1. Solubilization lex

(about

treated

120 mg)

with

and 0.13

5-6 ml at 0" for

saturation

collected

3 ml of Tris

The solution

showed

column.

(2.5

x 43 cm) of Llltrogel

tion

buffer.

1.6 ml/l0

min.

files

for

protein,

sults

of SDS polyacrylamide

which

peak was OP-C; and the

last

peaks

the

first

pattern at a position

are

electrophoresis,

with

shown

no.

corresponding 592

72

a coated

the

at a flow

1.

color

on re-

of the fractions the

in Fig. weight

second The SDS

protein.

to a molecular

of pro-

Based

c -1;

weight

solu-

rate

The elution

in Fig.

was shown

at pH

to a column with

cytochrome

peak was a low molecular of fraction

ammonium

with

was applied

collected.

of

was dis-

in an HPLC

on the

peak was mainly

volume

saturation

same buffer

1.6 ml each were

gel

fractionated

had been equilibrated

with

6.4% S-

(25 mM-50 a&0.5%)

the solution

heme and QP-C activity

the

band was observed

protein

comp-

pH 8.0 was

in a total

and 0.48

solution

major

M sucrose,

sulfate

was then 0.23

--b-cl-111

of 0.5% Lubrol-PX,

of ammonium

was eluted

and fractions,

electrophoresis

0.32

the presence

separation,

and the OP-C activity,

gel

containing

NaCl-Lubrol

AcA-44

The column

Cytochrome

between

three

For actual

of QP-C --

The mixture

in about

silica

buffer in

30 minutes.

The fraction

solved 8.0.

in 50 mM Tris

1.6 M guanidine-HCl

mercaptoethanol

sulfate.

and purification

2.

The main of 15,000.

Vol. 104. No. 2, 1982

Fig.

weight

molecular

nal

marking).

This

fraction

band

with

-cf.

was confirmed

A trace

point

of QP-C;

therefore,

2.)

may be easily

with

designed,

a total

and then

amounts

volume diluted

--b-cl-III

focusing

the approximate the yield

These

to 500 ul with

mixtures Tris-sucrose 593

and found (The

molecular

was at least

arrow

weight

of OP-C is

buffer

after

of the

of

complex

were

mixed

(0.05

M-O.25

M)

pH 8.0

incubated

one

shows

-- Aliquots

or the --b-cl-IV

were

detected.

95% pure.

c reductase

buffer

(inter-

to contain

was low and improvements

the product

complex

lysozyme

CI was also

pt! 3.6.

of QP-C in Tris-sucrose

of 200 ~1.

containing

cytochrome

of ubiauinone-cytochrome

pg of OP-C depleted varying

sample

at approximately

Although

2. Reconstitution

the

by isoelectric

an isoelectric

Fig.

with

of contaminated

amount

was analyzed

the position

30-60

RESEARCH COMMUNICATIONS

profile for the distribution of protein, heme and OP-C activity from Ultrogel AcA-44 column of a solubilized fraction of the Column size was 2.5 x 43 cm; flow rate of 1.6 ml/ b-c -III complex. iO-&n/fraction. The QP-C activity was estimated by incubation 40 ~1 elwte with the b-c -IV complex at 35" for 1 hour. The mixture was chilled and dilute a to 500 ~1. 10 nl aliquots were assayed for QH2-cytochrome 5 reductase activity at about 23".

The

method

AND BIOPHYSICAL

Elution

1.

15,000;

BIOCHEMICAL

at

35"

for

incubation.

one hour Ali-

in

BIOCHEMICAL

Vol. 104, No. 2, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

origin

89l-

6L5 =

4-

.s? z

3-

i

5 2 P

2-

Qp;C serum albumin corbonlc onhydrose and lysozyme

I

I

I

I

02

04

06

08

II

MobMity

Fig.

quots

2.

were

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of QP-C. The sample after treatment in 2%,SDS and 1% mercaptoethanol at 50" for 25 min was run on 10% acrylamide gel (5 mm inner diamter and 7 cm long) with 0.27% N, N' methylenebisacylamide cross-link. On the right of the figure are the drawings of gel patterns obtained after staining with Coomassie brilliant blue for the sample and the sample plus internal markers. The arrow on the left shows the position of OP-c.

withdrawn

cytochrome

to reconstitute

inactivity

molecular Calculating

ratio

We have weight

the

explanations

offered:

(a)

may have

a similar there

for

occurred

may be two QP-C's

system.

At present,

O. which

act

one,

its

we favor

on the protoplasmic

although

both

by QP-S

in SDS polyacrylamide

3, it

can be seen that

based to

on the molecular c+ greater

in the active

preparation;

may be present;

that

reaction, weight

QP-C.

SDH, as witnessed

the multiplicity

of QP-C

be a monomer

point

with

with

reported

in Fig.

than

during

isoelectric

reductase

of QH2-

of the

(9).

a ratio

QP-C may not

the restoration

by reconstitution

molecular

results

of QP-C to -1 c was more

Several

3 shows

complex

previously

proteins

from

Fig.

the succinate-DCIP

same monomeric

electrophoresis.

15,000

tion

the

assay.

succinate-Q

to catalyze

and QP-C possess gel

enzymic

of the --b-cl -IV

-c reductase

QP-C failed the

for

appear

(c) and

to be identical

the last

possibility.

and matrix 594

sides,

weight

than

unity

form; other

the molar

(b) 15,000

may be

some inactivaprotein

(d) as suggested in

the

More

with

before

--in vitro explicitly,

respectively

of 15,000.

(lo),

assay Qi and

(ref.

10 and

Vol. 104, No. 2, 1982

BIOCHEMICAL

1

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

1

I

I

13.6

27.2

40.6

54.4

QP-C hg 1 Fig.

3.

references

Reconstitution of ubiquinone cytochrome c reductase system contains 55 mg of the b-cl-IV complex and of QP-C-(fraction no. 68) as indicated in a toTaivolume of 200 ~1 Tris 50 mM pH 8.0 The mixture was incubated at 35" for 1 hour. After incubation, it was made up to volume 500 ~1 with 0.05 M Tris 0.25 M sucrose buffer, pH 8.0. 10 ~1 aliquots were drawn and QH2-5 reductase activity was assayed in 1 ml assay mixture in presence of 70 M Q2H2 at about 23'.

cited)

in

duct.

The orientation

tioned

so that It

these

was found

that

paramagnetic flavin

species

does not

show signal

EPR signal

only

Finally, garding separate teins.

the

the

the at least

isolation

point,

three

QP-C, recently

because

QP's.

one-direc-

in activity. complex

the

also

restores

at room temperature. signal

the --b-cl-IV

isolated

pro-

in the

is

due to any

complex

contains

undenatured

The Rieske

protein

form

(11)

no (11)

can show

temperatures. of QP-C eliminates of QP in the

of Q-bound

At this

than

isolated

be uniformly

complex that

at room temperature.

essentiality

class

in the

of QP-C to the --b-cl -IV of the --b-cl-111

protein

at low

may not

interchangeable

out any possibility other

and the Rieske

are

addition

rules

may exist

vesicle

two QP-C's

EPR signal

observation

Q cycle

in artificial

the ubisemiquinone This

the protonmotive

proteins

we have

respiratory

comparable

succeeded

questions

in

(*,

chain.

QP's

to heme proteins isolating

12,

13) re-

constitute and flavopro-

and purifying

two of

a

Vol. 104, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-- Technical assistance of Michael Seaman and Zhen-ping Acknowledgment This research is supported by grant6 of Zhang is gratefully acknowledged. NIH - HL 12576 and American Cancer Society BC-349. REFERENCES In New Horizon6 in Biological 1. King, T. E. (1980) Ubiquinone Proteins. Chemistry (A Festschrift to Professor K. Egi -- Koike, M., Nagatsu, T., Okuda, J., and Ozawa, T., eds.) 121-134, Japan Scientific Societies Press, Tokyo, Japan. 2. Yu, C. A., Yagoaka, S., Yu, L., and King, T. E. (1980) Arch. Biochem. Biophys., 204, 59-70. 3. King, T. E. (1981) Mitochondrial Coenzyme Q Linked Enzymes. In Biochemical and Clinical Aspects of Coenzyme Q (Folkers, K. and YamaKra, Y., eds.) 291-308, Elsevier/North Holland, Amsterdam, The Netherlands. 4. Yu, C. A., Yu, L., and King, T. E. (1977) Biochem. Biophys. Res. Commun., 2, 939-946, 5. Nagaoka, S., Yu, L., and King, T. E. (1981) Arch. of Biochem. and Biophys. 208, 6.

334-343.

Engel,

W. D.,

Acta.,

592,

Schagger,

H. and von Jagow,

G. (1980)

Biochim.

Biophys.

211-222.

7. Shunk, C. H., Linn, B. D., Wong, E. L., Wittereich, P. E., Robinson, F. M., and Folkers, K. (1958) .I. Am. Chem. Sot., 80, 4753. 8. Wei, Y. H., Scholes, C. I'., and King, T. E. (1981) Biochem. Biophys. Res. Commun.. 2, 1411-1419. Y. L. (1975) An Examination 9. King, T. E., Yu, C. A., Yu, L., and Chiang, Mechanisms and their Uncertainties Involved of the Components, Sequence, in Mitochondrial Electron Transport from Succinate to Cytochrome c. In Electron Transfer Chain6 and Oxidative Phosphorylation (Quagliari&oy E Papa, S., Palmieri, F., Slater, E. C., and Siliprandi, N., eds.) li;-118, ElsevierjNorth Holland, Amsterdam, The Netherlands. IO. Kim, C. H. and King, T. E. (1981) Biochem. Biophys. Res. Commun., 101, 607-614. 11. Trumpower, B. L. and Edwards, C. A. (1979) J. Biol. Chem., 254, 8697-8706. 12. Rich, P. R. and Bendall, D. S. (1979) FEBS Lett., 105, 189. 13. Hatefi, Y. and Galante, Y. M. (1980) J. Biol. ChemTE, 5530-5531.

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