Reconstitution of succinate-Q reductase

Vol. 79, No. 3, 1977

BIOCHEMICAL

RECONSTITUTION Laboratory

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF SUCCINATE-Q REDUCTASE

C. A. Yu, L. Yu, and Tsoo E. King of Bioenergetics and Department of Chemistry State University of New York at Albany Albany, New York 12222

October

31,1977

SUMMARY--Reconstitution of succinate-Q reductase is achieved by admixing soluble succinate dehydrogenase (SDH) and ubiquinone-protein-S (QP-S), a new protein isolated from the soluble cytochrome The reconsti--b-cl complex. tuted reductase catalyzes reduction of Q by succinate. The reaction is fully sensitive to thenoyltrifluoroacetone. The reconstituted reductase (same as succinate-cytochrome 2 reductase or submitochondrial particles) does not show "low concentration ferricyanide reductase activity" as soluble dehydrogenase does. In other words, this enzymic site on SDH is occupied by QP-S. When an artificial dye, such as phenazine methosulfate or Wurster's Blue, is used as electron acceptor the rate of oxidation of succinate by SDH is not significantly changed regardless of whether the dehydrogenase is in the free or in the reconstituted succinate-Q reductase forms. Freshly

prepared

genase

possesses

quires

low

ferricyanide

concentrations

tion

exists

iron (5)

-

(Km s 10 mM) (2,

activity

Blue

phenol

(1)

the action

but not

inhibits

sulfide

(Q)l,

succinate 5 reductase

oxidation

a correlalow-fer-

(high

potential

(i.e. --

Center

use phenazine

electron

acceptors. (TTFA)

enzyme but markedly catalyzed

as well

that

needs

dyes

S-3) (6,

7)

or 2,4-dichlorophenolindo-

thenoyltrifluoroacetone soluble

type center

can also

as artificial

of the

the other

reconstitutivity,

of the HiPIP iron

dehydroOne re-

has been claimed

parameters:

ubiquinone

among others,

activities

succinate-cytochrome 1

(8),

succinate

activities.

m ?J 50 PM), while It

dehydrogenase

of inhibitors,

the artificial quantities)

3).

and the appearance

succinate

and Wurster's

to

(K

three

active

reductase

as shown by EPR technique)

In addition,

(DCIP)

following

(4)

(1)

ferricyanide

concentrations

among the

protein

reconstitutively

two distinct

high

ricyanide

soluble,

as succinate

In regard

barely (in

by reconstituted oxidase

inhibits micromolar or intact

(see Fig.

1 of

Abbreviations used: DCIP, dichlorophenolindophenol; HiPIP, high potential iron protein; PMS, phenazine methosulfate; Q, ubiquinone; QP-S, ubiquinonebinding protein in the succinate dehydrogenase region; SDH, succinate dehydrogenase; TTFA, thenoyltrifluoroacetone; and WB, Wurster's Blue.

Copyright 0 1977 by Academic Press, Inc. AN righrs of reproduction in any form reserved.

939 ISSN

0006-291X

BIOCHEMICAL

Vol. 79, No. 3, 1977 Ref.

1).

with

It

is also

half-lives

we have

binding

(hydrogen)

reports

reconstitution the locus

differences

and 8 hours, in

demonstrated (QP-S,

carrier

previously

from

exists

in the

unstable

reconstitutive

(1). of a ubi-

which

to ubiquinone

and other

can serve

(9).

This

properties

delineating

are

also

In addithe

and dehydrogenase

chain,

as an

paper

from QP-S and SDH.

dehydrogenase

MATERIALS

of air

QP-1)

reductase

respiratory

for

and isolation

called

succinate

succinate

enzyme is

respectively,

the existence

of TTFA inhibition free

the soluble

the presence

of succinate-Q

between

or as it

that

at O-4"

protein

electron

QP-S,

0.5

activities

Recently

tion,

documented

of about

and artificial

quinope

well

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bound

to

reported.

AND METHODS

Succinate dehydrogenase (1, 7), the cytochrome b-51 complex (lo), the heart muscle preparation (ll), and Wurster's Blue (12) were prepared by previously reported methods. QP-S2 was prepared as briefly described in (9); details will be published elsewhere. The concentrations of electron acceptors were measured spectrophotometrically and the millimolar extinction coefficients (7, 12) used were 5.2 at 510 nm, 21 at 600 nm, and 1.0 at 410 nm, for Wurster's Blue, DCIP, and ferricyanide, respectively. Enzymic assays were conducted spectrophotometrically at room temperature, about 23". The reaction mixture, in a final volume of 1 ml, contains 50 mM Na-K phosphate buffer, pH 7.8; 1.5 mM KCN; 1 mg/ml bovine serum albumin; 40 mM succinate and various amounts of proper electron acceptors (cf. Ref. 7). It must be mentioned that cyanide was added by following the conventional method (see Ref. 13 for easier comparison of data appearing in earlier literature), rather than for the possible contamination of oytochrome oxidase. Indeed, all QP-S and SDH described in this paper are completely free of any cytochrome. Spectrophotometric measurements were done in a Cary spectrophotometer, model 16, with a recorder. RESULTS AND DISCUSSION Figure activity

of freshly

K3Fe(CN)6 of the

at 23".

tracing,

When this about

1A shows

the actual prepared

tracing SDH.

The specific is

preparation

13 umoles3

of the

The assay was conducted

activity,

as calculated

succinate

was simply

oxidized

admixed

5 mol QP-S per mol of SDH) and then

2 A part of Q of the and purification.

QP-S isolated

low-ferricyanide

with

with

excess

amounts

under

the

slope

mg protein. of QP-S (i.e. -same conditions

in the course

3 The range of activity is usually between 12 and 16 umoles oxidized per min per mg of SDH at room temperature.

940

350 uM

by the initial

per min per

assayed

has dissociated

reductase

of isolation

of succinate

Vol. 79, No. 3, 1977

Fig.

1.

no activity Q (Fig.

succinate-Q reductase is

(< 5%) of free

1A);

reductase activity

further

SDH.

ductase

activity

rapidly

in air

indicates

was stimulated unlike

does not (cf. -

Ref.

1).

to that

of the

functionally particles

(cf. not

only

Ref.

enzyme, time;

The slower

--l b-c 14).

reconstitute

complex

rate

(L.e. (10)

of

converted

to

ferricyanide

of succinate-Q

reductase

of Q, the reduction

as that

in the initial

free

rate

succinate-Q

re-

SDH inactivates

shown at the end of the trac-

QP-S bound or alkaline h-cl

SDH to form

941

the absence

low

of ferricyanide

The cytochrome with

so-called

reconstituted whereas

of exhaustion

of QP-S toward

cytochrome

same level

in

now being

upon the addition

decay with

ing was due to the approach The reaction

that

free

of the Formation

to the the

SDH is

site

occupied.

by the fact

Also,

SDH was observed that

and the active

evidenced

of free

this

of SDH is

of ferricyanide

mixture.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Activities of succinate-dehydrogenase and reconstituted succinate-Q reductase. (A) Ferricyanide and (B) Wurster's Blue were used as electron acceptors: 5 ~1 of freshly prepared SDH, 0.77 mg/ml, were used in the assay. In the reconstituted system, excess QP-S (5-fold of the dehydrogenase) was mixed with SDH; the concentration of SDH was also adjusted to 0.77 mgfml.

practically exogenous

BIOCHEMICAL

complex

in the reaction to)

SDH is

treated

particles

or alkaline

succinate-cytochrome

identical

treated c re-

BIOCHEMICAL

Vol. 79, No. 3, 1977

Fig.

2.

Double reciprocal reductase activity for the enzymes The concentrations Blue (B) in the concentration of the PMS systems

ductase

or succinate

ferricyanide

plots for SDH and reconstituted succinate-Q against acceptor concentrations. The conditions used were identical to those detailed in Fig. 1. of the immediate acceptors of PMS (A) and Wurster's reaction mixture are given in the plots. The DCIP in (A) was kept at constant, 45 mM. In all described in this paper, the final acceptor was DCIP.

oxidase,

reductase

respectively,

activity

by the able

fact

lB, that

to catalyze

dye reduction ductase;

Figure succinate-Q

free

reduction

the rate not

Q also

affected double

reductase

activities it

by the

resulted

formation

reductase

by SDH in

of free

decrease

significant

942

complicated are of this

of succinate-Q

SDH'and

WB and PMS assay

in a slight

succinate not the Vmax . In the PMS system V succinate was observed in reconstituted lMX

were

as

re-

the presence

of Q.

plots

toward

results,

The stimulation

of WB catalyzed

reciprocal

the low

similar

the results

15).

the

addition

abolish

succinate-Q

Ref.

confirms

of reduction

2 depicts

was used as acceptor

acceptor,

case

of WB (cf.

also

same time.

electron

In this

they

SDH and reconstituted

by exogenous

is

as the

observed.

both the

whereas

of succinate

were

but

of SDH at the

When WB (260 uM) was used shown in Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

succinate-Q

systems.

in Km for increase

reconstituted

both

reductase.

When WB the dye but PMS in Km and It

should

be

Vol. 79, No. 3, 1977

TABLE I.

BIOCHEMICAL

Km and "max of Succinate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dehydrogenase

and Reconstituted

Succinate-

Q Reductase. Electron Preparations

Succ.. + PMS-DCIP V

Km Succinate

VIIBX

0.3

18

reductase

0.9

22

is

expressed

temperature

mentioned less

artificial

cinate-Q

reductase

It

is

ase is

known

drial

particles,

acceptors

using that

but

not

enzyme for

shows the inhibition

90% of activity

just

like

the enzyme bound

ly unaffected

which

identical

behavior

1 of Ref. to that

succinate

oxidation

cytochrome

form which (1).

the

0.7

41.7

at room

to locate

of SDH and suc-

when

reacts

only

artificial reductase

the TTFA locus.

On the other dehydrogenase

Reconstituted

or submitochon-

with

Figure

reductase

by TTFA in micromolar

free

the dehydrogen-

succinate-Q

succinate-Q

hand, activity

of inhibitor succinate-Q

of Q.

of Q present

-c reductase

Reconstituted

the concentration

of free

41.7

of Q regard-

to the amount

acceptors.

to particles.

1).

1.1

by the presence

electron

was inhibited

measures

affected

of Km and V,,,

in soluble dyes

V max

In the case of succin-

the data

such as in

by TTFA even if

as 3 mM (See Fig.

is used.

of TTFA of reconstituted

than

by the presence

proportionally

experimentation

More

to PMS reaction

affected

to be greatly

TTFA inhibits form"

21.6

per mg SDH per minute

acceptor

different

such as phenazine

is an ideal

not

I summerizes

the ucomplex

in

SDH is

electron

Table

Km

in mM.

of the dye decreases

the system.

Succ. + WB

VIllaX

Km

0.035

of dye reduced expressed

K PMS was found m

reductase,

The Km value in

Km is

PMS the Km for

that

of which

ate-Q

as umoles

(23').

Succ. + Q-DCIP

max

dehydrogenase

Succinate-Q

Acceptors

activity.

concentration the succinate was completeused was as high reductase

showed

SDH when PMS was used as the electron

943

3A

Vol. 79, No. 3, 1977

Fig.

3.

BIOCHEMICAL

Inhibition of thenoyltrifluoroacetone of reconstituted succinate-Q reductase activity. (A) was the titration of TTFA using Q (15 mM) and PMS (1.8 mM) as electron acceptors, and (B) was the double I reciprocal plot of succinate-Q reactivity in -vs. Q concentration the presence and absence of TTFA.

acceptor.

When reconstituted

concentrations inhibition

of Q in was observed.

Figure

of Q and the

the inhibitor.

An inhibitor

should

refer

be mentioned

to only

Recently, the enzymic Vinogradov

Ackrell tion resolve

all

(8)

using

--et al.

found between

that

acceptor

various

competitive plots

of the

and absence to be around

of 30 ).M.

of Q and TTFA used here of the solubility than

these

of the

WB and PMS systems

as that difference

two dye systems.

regarding

for

oxidation using

re-

values.

8) have appeared

SDH catalyzes

no significant these

was calculated

(16,

under

reciprocal

in the presence

be smaller

articles

reported

the double

Because

might

WB as electron

(16)

of succinate

Ki,

Vmax, of SDH using

et al.

was assayed

the concentrations

two controversial

as fast

activity

constant,

concentrations

assay.

of succinate PMS; whereas,

in the rate Our results

of oxidaautomatically

the disparity. On the other

effect

3B shows

concentration.

activity,

reductase

of 10 pM and 56 nM TTFA,

enzymic

that

apparent

the true

agents,

twice

succinate-Q

the presence

concentration

It

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of "membrane

hand,

our

results

environment"

give on SDH.

944

no support Actually

to the claim the

so-called

of the "membrane

Vol. 79, No. 3, 1977

is

environmentv dehydrogenase amount

BIOCHEMICAL

just

no more than

combines

with

possibility

for

structure is not

likely.

plained cinate

QP-S is

"membrane

environment"

In conclusion, with

well

"membrane

defined

in

for

environmentn

inhibition

and the reaction

themselves

cannot

directly

rate

arguing

of reconstitution

SDH and QP-S not but

its

acceptor

than

only

a small the

in a "membrane-like" recombination

with

SDH

of the dyes can be exand thus for

oxidized

suc-

the requirement

of a

such a reaction.

the success soluble

after

electron

succinate

QP-S contains

reductase

(17)

way rather

after

was made in detergent,

the reduction

the native

in a more effective

isolated

of succinate-Q

environment"

The increase

since

Since

exhibited

and the protein

the formation

of a "membrane

the behavior

QP-S.

(< 20%) of phospholipid4

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

also

only

unequivocally

locus interact

of DCIP (or with

of succinate-Q resolves

reductase

the "mystery"

defines

the sites

of TTFA

related

compounds)

which

of

SDH.

ACKNOWLEDGMENTS Health

This work was supported by grants from the National and the American Heart Association, Northeastern

Institutes New York

of Chapter,

Inc.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

King, T. E. (1966) Adv. Enzymol. 2&, 155-236. Vinogradov, A. D., Gavrikova, E. V., and Goloveshkina, V. G. (1975) Biochem. Biophys. Ras. Commun. 65, 1264-1269. Kettman, J. R. (1967) Ph. D. Thesis, Oregon State University, Corvallis, Oregon. Vinogradov, A. D., Ackrell, B. A. C., and Singer, T. P. (1975) Biochem. Biophys. Res. Commun. 67, 803-809. King, T. E., Ohnishi, T., Winter, D. B., and Wu, J. T. (1976) in Iron and Copper Proteins (Yasunobu, K. T., Mower, H. F., and HayaisK, O., eds.) pp. 182-227, Plenum Publishing Corp., New York. King, T. E. (1963) J. Biol. Chem. 238, 4032-4036. King, T. E. (1967) Methods Enzymol. lo, 322-331. Vinogradov, A. D., Goloveshkina, V. G., and Gavrikova, E. V. (1977) FEBS Lett. 12, 235-238. Yu, C. A., Yu, L., and King, T. E. (1977) Biochem. Biophys. Res. Commun. 78, 259-265. Yu, C. A., Yu, L., and King, T. E. (1974) J. Biol. Chem. 249, 4905-4910. King, T. E. (1967) Methods Enzymol. 1.0, 202-208.

4 Accurate

determination of lipid requires large amounts of the sample, thus 20% is the maximal value. More likely it is much smaller than the cited value when sufficient sample is available for assay.

945

Vol. 79, No. 3, 1977

12. 13. 14. 15. 16. 17.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

King, T. E., Howard, R. L., Kettman, J., Jr., Hegdekar, B. M., Kuboyama, M, Nickel, K. S., and Possehl, E. A. (1966) a Flavin and Flavoproteins (Slater, E. C., ed.) pp. 441-481, Elsevier Publishing Co., Amsterdam. Bemath, P., and Singer, T. P. (1962) Methods Enzymol. 5, 597-614. Keilin, D., and King, T. E. (1960) Proc. Roy. Sot., London, Series B, 152, 163-187. Mustafa, M. G., Cowger, M. L., Labbe, R. F., and King, T. E. (1968) J. Biol. Chem. 243, 1908-1918. Ackrell, B. A. C., Coles, C. J., and Singer, T. P. (1977) FEBS Lett. 75, 249-253. Ackrell, B. A. C., Kearney, E. B., and Singer, T. P. (1977) J. Biol. Chem. 252, 1582-1588.

946