Carboxins: Powerful selective inhibitors of succinate oxidation in animal tissues

BIOCHEMICAL

Vol. 71, No. 1,1976

CARBOXINS:

POWERFUL

SUCCINATE P.C.

Biology

SELECTIVE

OXIDATION

Mowery,

Molecular

AND BIOPHYSICAL

IN

B.A.C.

ANIMAL and

Veterans

OF

TISSUES T.P.

Singer

Administration

San Francisco, California 94121 and Biophysics, University of California,

and

INHIBITORS

Ackrell,

Division,

RESEARCH COMMUNICATIONS

Hospital,

Department of Biochemistry San Francisco, California

94143

and G .A. Agriculture

Received

White

and

Canada, Research London, N6A

G D . Thorn

Institute, University 387 I 0 ntario, Canada

Sub

Post Office,

May 14,1976

Summary: Carboxin (5 ,bdihydro-2-methyl1,4-oxathiin-3-carboxanilide) is a systemic fungicide, reported to inhibit succinate oxidation in certain fungi, particularly Ustilago ma dis (corn smut). In the present study the action of carboxin and of other oxa+- erivatives on beef heart succinate dehydrogenase has been investigated. Carboxins inhibited the same activities to the same extent as thenoyltrifluoroacetone (TTF) but at much lower concentrations. For 14 carboxin derivatives the inhibition constants (concentration required to inhibit 5% of the carboxin-sensitive activity) ranged from 2 x lo-* to 2 x 10-6,M. Like TTF, carboxin derivatives did not inhibit soluble succinate dehydrogenase butinhibited the reduction of coenzyme Q analogs, of 2,6-dichlorophenolindophenol, and of phenazine methosulfate (PMS) in Complex II preparations. The same reactions and succinoxidase activity were also inhibited in inner membranes (ETP). In ETP only % 50 % of the succinate-PMS activity was carboxin sensitive, the same fraction as is’inhibited by TTF or is lost on extraction of coenzyme Q and on incubation with cyanide. While the inhibition of PMS reduction by carboxin was largely or entirely competitive in Complex II, it was predominantly non-competitive in ETP at low concentrations. Some other carboxin derivatives gave mixed inhibition patterns for PMS reduction in ETP even at low inhibitor concentrations. The complex inhibition pattern in the PMS assay seems more compatible with conformation changes affecting activity than with loss of a reaction site for PMS .

Carboxin nuda

and

Rhizoctonia

cornsmut

(U. -

denitrificans

the

genetic the

Copyright All rights

solani

have

oxidation primary

(1).

inhibition

shown is in the

reaction

studies

a striking

fungicide

maydis)(2,3), (4)

succinate that

is a systemic

with

with

yeast

that

the

to the

0 1976 by Academic Press, Inc. in any form reserved.

of reproduction

(3),

and

membranes

by carboxin

of the

of U.

354

maydis

paper

in Ustilago

fungi,

for blocking

chain.

II region (5).

Further has come

The

preparations describes

particularly

Micrococcus

responsible

respiratory

in fungal

The present

metabolism

from

of carboxin

is in the Complex mutants

of TTF.

succinate carboxin-sensitive

with site

II region

of carboxin

action

with

inhibition

Complex

oxidation

to inhibit

Investigations

carboxin-resistant

of succinate similarity

site

known

evidence from

characteristics (2,3) the

of

suggested

inhibition

of

VoL71,No.

beef

1,1976

heart

ranging

BIOCHEMICAL

succinate from

dehydrogenase

inner

membranes

AND

BIOPHYSICAL

by a series (ETP)

to the

MATERIALS

RESEARCH

of carboxin

purified, AND

COMMUNICATIONS

derivatives

soluble

in preparations

enzyme.

METHODS

ETP and Complex II and reconstitutively active, butanol extracted succinate dehydrogenase were isolated as previously described (6-8). Activities were measured in fully activated preparations: succinate-PMS reaction with DCIP as terminal acceptor (9), DCIP reduction under the same conditions but without PMS present, succinoxidase polarographically. The reduction of the Q analog, DPB (2,3-dimethoxy+methyl-6-pentyl-1,4benzoquinone) was measured with DCIP as terminal acceptor under the conditions of the PMS-DCIP assay (9) but with 39 uM DPB in lieu of variable concentration of PMS and “low K ” ferricyanide activity (l~spectrophotometrically. DPB was obtained from Dr. Karl Fon
of the succinate-DPB --

known

tives

by succinate

with

DPB,

succinate while Linear

for

many

in Complex

which

II (12).

plots, carboxin

Complex

produced indicating and

DCIP

reductase

activity

three

inhibitors

produced

succinate-DCIP

TTF

completely present

Q,

in this

II was

~95%

comparable saturation

TTF,

1.5

of Complex complete

to

study assay

inhibition

reduction

The

highly

with

of Q deriva-

0.3

non-competitive

assayed

of DPB

concentration. Ki uM-

values

at 15O

The

succinatc-

.

to inhibition. kinetics

Jii

and

All linear

e

24.0

.

200

2

160

12.0

00

.

40

0

0

mw

20

40

6.0

PMS

Fig. 1. Effect of 3’-methylcarboxin on membrane-bound succinate dehydrogenase. Ordinate, unu les of succinate oxidized/min/mg of protein at 38’. A. ETP (turnover number = 19,400 at 38O); inhibitor concentrations: 1, none; 2, 0.05 uM; 3, 0.15 @; 4, 1.5 uM; 5, 5.0 uM. 8. Complex II (turnover number= 10,800); T no inhibitor; 2, 0.84 3 3’-methyTarboxin.

355

by

TTF or carboxin,

giving

sensitive

was

reduction

at 7 to 10 uMobtained,

in Complex

activity

at 60 uM -

3’-methylcarboxin,

II was similarly

activities --

Q reductase

inhibited

were

:JM; -

the

(13).

inhibition

kinetics, 1.9

reductase inhibits

In the

replaces

- activated

3’ -methylcarboxin

as follows:

that

quantitatively

in NO;

Dixon

years

and -

II.

Vol.

BIOCHEMICAL

71, No.

1,1976

plots.

The

Dixon

methylcarboxin,

and

petitive

and

with

calculated

for

number

observed JO,OOO).

and

cyanide

branes.

(15)

All

value

these

(*

A number

10,000)

were

Ki

values

different

from

As shown at 15’

in typical

Complex

in Complex

oxidation

effect however,

inhibition

lead

of these from

compounds that

observed

was non-competitive

point 2),

a competitive the

same

as with

to loss of about

treatments

reduce

seen

in typical

the

derivatives

Titration of succinate-PMS (A) at 38’. The ordinate

appeared

TTF.

Extraction

of the

turnover

have

number

concentrations DPB,

1A).

of ETP ( s 20,000

and in

of Complex

high

activity

of

reaction

samples

of Q10

numbers

II

concentraMaximal

(14) in

or incubation inner

mem-

+ 1,000)

to

II preparations. been

compared

CONCENTRATION,

activity represents

turnover

succinate-PMS

PMS-DCIP

3,‘-

a turnover

of DCIP,

(Fig.

uIvJ;

those

having

low

in typical

feature

1.1

to

reduction

up to a relatively

half

Complex

on the

is com-

similar

higher

reduction

in the

of PMS

(preparations

the

II,

inhibition

TTF,

inhibition

In ETP preparations

inhibited

in Complex

the

pIvJ;

3’-

Carboxin,3’-

very

with

PM; -

observed

lA,

to 1.5

II samples

in -- ETP.

completely The

of carboxin

II preparations

1.1

II.

nevertheless

of the

below,

TTF,

pattern

in Fig.

were

COMMUNICATIONS

by succinate the

character

INHIBITOR

Fig. 2. carboxin

of PMS

competitive

.

(Fig. also

reduction

PM; -

in Complex

The

PM -

at which

was ~50%

activity

1.4

different,

of inhibitor,

the

to 0.9

carboxin,

3’-methylcarboxin

In ETP the

0.7

RESEARCH

reduction:

activity.

1B).

The

As shown

markedly

carboxin,

reactions.

for DCIP

of succinate

inhibition

the

inhibition

Inhibition

ETP was

with

of the

was observed.

succinoxidase

tion

inhibited

and

BIOPHYSICAL

reductase

also

inhibition

carboxin

were:

succinate-PMS

to PMS.

0.3

of

15”

succinate-DCIP

DPB

regularly

(Fig.

PM. -

llF

respect

methylca&oxin,

mixed

to 0.4

characteristics

succinate-DPB

was

at

of -- the

methylcarboxin, the

values

0.3

Inhibition

but

Ki

AND

for

in inhibiting

,uUM

of ETP with carboxin Vmax (PMS) , based

356

effectiveness

(0) and 3’-methylon the data of Fig.

1A.

Vol.

71, No.

BIOCHEMICAL

1,1976

24.0

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

t

012345 [PMS,

Fig. 3. turnover

the

Effect number

of 4’-phenoxycarboxin of the ETP used

succinate-PMS

competitive tive

reaction

kinetics

inhibition

attained

(Fig.

and

was

in the

In comparing from

Dixon

thus

the

plots

when

inhibition

(12,,.)

and

particular

compound

curved

scale

of potencies

a part

of the

measured

all

Dixon

plots.

however

Some (Fig.

inhibitors

(Table

I inear.

In addition,

the

50?? (Table

by modifying

is susceptible

of them

showed

non-

produced

The

maximal

1A.

uncompetiinhibition

I).

carboxins,

giving

of them

3).

of various

calculated

activity

most

potency

concentration

is possible

activity of ETP at 38’. The concentrations were as in Fig.

concentrations

plots,

for

were

were

low

linear

inhibitory

( Iso)

lative

At

Dixon

of 5&b

these

the

ETP.

1A) and

range

on succinate-PMS 18,000; inhibitor

was

in

mM]-’

Ki

values

concentrations

loss of the I).

Another

the

Hill

activity

were

calculated

giving

25%

inhibitable

basis

by

for establishing

equation

to fit

cases

the a re-

where

only

to inhibition: zE - T

log K - nlogi=log

z(T-E)’ where

T is the

particular of the and

activity

inhibitor fraction

of the

of inhibitor-resistant to last

As seen

in Table

methods inhibitors

different gave than

column) I, from

gratifyingly TTF

and

with the

inhibition

apparent

in Table

exceptions

carboxin

Ki values

ranged

357

for

n (a measure

the

K values

determined

All

values.

and

presence

of a

z the

reciprocal

of cooperativity)

I.

where

constants

in the

of inhibitor,

Values

included

three

similar the

concentration

activity. are

E the activity

enzyme,

i the

concentration,

of K (next

significantly

uninhibited

by other

derivatives from

from

plots

means,

were

2 x lOmE

Hill

more

to 2 x 10

were

the -ytent - M.

four

Vol.

71, No.

BIOCHEMICAL

1,1976

AND

BIOPHYSICAL

TABLE COMPARISON AND

OF

OF

THE

POTENCY

SUCCINATE

OF

Inhibitor

assay

cC oncentration plot.

eKi

reported t ions

at 38’

from

from

Dixon

TTF but

heart.

of PMS-DCIP

OF

nd

ETPa

Kd

Ke

(IJ~

(lg

9.0 3.0

9.6 2.6 1.5

57 57 55

1.00 0.89

9.7 2.4

0.53

1.4

0.90 0.85 0.66 0.55 0.36 0.14 0.08

0.80 0.70 0.70 0.60 0.33 0.15 0.06

47 47 52 52 47 51 44

0.92 0.88 1.30 0.64 1.40 0.88 1.50

0.75 0.72 0.60 0.61 0.22 0.18 0.02

0.78 0.80

0.06 0.04 0.03 0.03 0.02

0.09 0.05 0.04 0.02 0.02

58 52 54 39 51

0.76 1.20 1.10 1.50 1 .Ol

0.16 0.03 0.03 0.002 0.02

0.11 0.05

for

of carboxin

25%

sensitive

inhibition

0.50 0.14

of control d

activity.

K and

activity.

n values

from

plot.

on purified --not

In our

succinate

carboxin hands

blocks neither

or of ferricyanide

dehydrogenase.

Schewe

the succinate-PMS

TTF (“low

nor

carboxin

Km”

site

reaction (6 uM) -

(10))

et al. --

( 16)

in soluble

blocked

prepara-

either

the

in a reconstitutively

reduc-

active,

preparation. Effect ----

succinate

of carboxin

on succinate

dehydrogenase

particles

( 17).

Ki values

on succinate

dehydrogenase

aerobic

yeast and

in submitochondrial

of 0.54 to be

from

3’-Oxtoxycarboxin

non-competitively

appear

ACTIVITY

7.5 1.9 1.3

bConcentration

inhibition

of carboxins

that

soluble

(V max).

for 5oo/o

--Effect

tion

DERIVATIVES

Max.

‘PMS-DCIP

Hill

CARBOXIN

DEHYDROGENASE

TTF 4’-D imethylaminocarboxin 4’-Carboethoxycarboxin 5,6-Dihydro-2-methyl-1,6oxathiin3-carboxanilide (Carboxin) 4’-Benzoylcarboxin 4’-n-Butoxycarboxin 3’-nethylcarboxin 4’-Phenylcarboxin 4’-n-Butylcarboxin 4’-Fhenoxycarboxin 5,6-D ihydro-2-methyl1 ,boxathiin3-n-decylcarboxamide 3’-Plienoxycarboxin 4’-n -Pentylcarboxin 3’-dctoxycarboxin 4’-Hexoxycarboxin

COMMUNICATIONS

I

INHIBITORY

TTF TOWARD

RESEARCH

uM -

and

3.9

@,

is 5%

to the

yeast

dehydrogenase

is qualitatively

than

were

by

linear

reduction

the

Dixon

enzyme,

although

two

yeast

plots, Thus

in the

by

TTF in submitochondrial

calculated.

to the heart similar

PMS

inhibited

giving

respectively,

yeast.

inhibited

4’-hexoxycarboxin particles,

less inhibitory

from --

sources.

enzyme from

which

carboxins their

action

BIOCHEMICAL

Vol. 71, No. 1,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION The

results

succinate

presented

dehydrogenase

manner

similar

maximal

inhibitions

the

functioning

come

from

gz1.94

reached

The

the

kinetics

ETP)

low

concentrations

tion,

as reported

the

give

respect

Hipip

II preparations

turnover

as has also by

3’-methylcarboxin number

declines

( 14), been

purely

a modified shows to 10,000,

and

yeast

affected

chelator

TTF,

Support

inhibit

are 1,000

of

in a and

the

interfere

for

this

the

reoxidation

the

latter)

complex.

with

idea

has

of both but

cause

at 38’

are

in the

the

exten-

(Fig.

is always such

inhibition

by TTF

procedure,

PMS-DCIP

assay, inhibi-

however: 3).

At

evident.

a few

relatively

In particles

in which

Complex

II samples

carboxins

is purely

competitive

(14).

yielding

reached

It is suggestive

that

a turnover

number

componentgo

that

on cyanide

Fig. 4. Comparison of cyanide inactivation and inhibition succinate-PMS activity in ETP and Complex II. Incubation for 1 hr at 30“, pH 8 .O; 3’-methylcarboxin, where present, turnover number = 19,000 at 38O. 8. Complex II, turnover

carhigh

as typical

a non-competitive the value

membranes

non-competitive

exceptions,

concentrations

with

In intact

or predominantly There

Q 10,000,

observed

iron

(particularly

+

TTF.

the

reactions

enzyme. only

component to

the

reduction

at low

is decreased

isolated by

( 14) for

tissues

operation

II preparations.

of 20,000

inhibition

membranes

not

enzyme

give

normal

TTF.

of the

of PMS

the

animal The

like

in Complex

carboxins et al. --

as with

carboxins

a competitive

number

to PMS,

center

number

by Rossi

same

of the

inhibition

of most

(2,3,18).

carboxins,

that

inhibit

from

fungi

component

centers

a turnover

inner

inhibition

Fe-S

Fe-S

concentrations

turnover

that

(13)

of the

with

the

(18)

uncompetitive

Q-depleted

the

Hipip of the

(e.g.,

carboxin

suggested

preparations

in other were

the observations

destruction

boxins

action

of a specific

and

derivatives

in membrane

to their

It has been

sive

showthatcarboxin

(8)

or with

in Complex of

Q 13,000,

at Vmax

treatment

by 3’-methylcarboxin with cyanide (0.02 was 0.84 uM. A. number = l-00.

(PMS)

(Fig.

4).

of M) was FI’P,

Vol.

71, No.

The

fact

by cyanide depleted lat (a)

BIOCHEMICAL

1,1976

all

that

inhibition

lower

the

or cyanide

‘ma* These

(PMS))

is not

readily

II (TN (b)

either

the

It has been

shown

number

succinate-PMS would have causes with respect leads

(19)

by

known, to complete

that

by

pool

of the

modification

these

inhibitors

interruption

the

idea

Fe-S

by

to it.

PMS

suggest

with

component

enzyme

action

of carboxins

might

likewise flux

which seems

bring via

about Q

and

Nelson

in Complex limiting.

the membrane

et al. A-

its interaction

of TTF

a conformational

in every

change

reactivity

(20)

dehydrogenase,

alters that

of the

to which

of succinate

profoundly

lowered

even

to be rate

access

all

hypothesis

a potentiation

(c)

to mimic

sites

Q , since

into

Q ( 14)),

cyanide.

carboxins.

reaction

that fast

Q-

or carboxin

two-site

enzyme

as by TTF and

in the

two

The

is too

may

that

of TTF and

showing

(possibly

an Fe-S

by TTF

of the

soluble

This

and

is endogenous

however,

of the

component

of electron

access

centers

as 50%.

one

inactivation

$ 10,000

of action

( 15),

( 13),

and

further

mode that

or block

on incorporation

as well

inhibited

the

COMMUNICATIONS

of Q,

in ETP to

activity

EPR data

on interacting

Since

the

measured

a membrane

- b.

for

with

by as much

carboxin,

TTF,

a conformational cytochrome

recent

increases

that

the Q

reoxidation

activity

be blocked proposed

with

of the

not

RESEARCH

depletion

enzyme

are

explanations

compatible

deplete

reconciled

of the

preparations

for ‘L 50%

BIOPHYSICAL

or by TTF,

number

several are

= 10,000)

its turnover

turnover

suggests

of PMS , responsible treatments

carboxins

inactivated

observations

these

by

AND

which

towards

PMS.

Service

(HL

ACKNOWLEDGEMENTS

16251)

I. 2. 3. 4. 5. 6. 7. 8. 9. 10.

This investigation and the National

was supported by grants from the U.S. Science Foundation (PCM 76-03367).

Public

Health

REFERENCES Mathre, D.E. (1970) Phytophatholgy, 60, 671-676. White, G.A. (1971) Biochem. Biophys.es. Communs. 44, 1212-1219. Ulrich, J.T., and Mathre, D .E. (1972)J. Bacterrol. 1% 809-817. Tucker, A.N., and Lillich, T.T. (1974) Antimicrobial Agents and Chemotherapy 6,572-578. Georgopoulos, S .G., Alexandri, E., and Chrysayi, M. (1972) J. Bacterial. - 110, 809-817. Ringler, R.L., Minakami, S., and Singer, T.P. (1963) J. Biol. Chem. -238,801810. Baginsky, M.L., and Hatefi, Y. (1969) J. Biol. Chem. 244, 5313-5319. King, T.E. (1967) Methods in Enzymology 10, 322-331.Singer, T.P. (1974) in Methods of Biochemical Analysis (Glick, D., ed.)g, 123175, J. Wiley, NewYork. Vinogradov, A.D., Gavrikova, E.V., and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Communs. 65, 1264-1269.

360

Vol. 71, No.

11. 12. 13.

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20.

1,1976

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

White, G .A. and Thorn, G .D . (1975) Pesticide Biochem. Physiol . 5, 380-395. Ziegler, D .M. (1961) in Biological Structure and Function (Goodwin, T.W., and Lindberg, 0. eds.) II, pp. 253-264, Academic Press, New York. Singer, T, P., Beinert, H . , Ackrell , B .A .C., and Kearney, E .B. ( 1975) in Proceedings of the 10th FEBS meeting (Raoul, Y., ed.), pp. 173-185, Elsevier, Amsterdam. Rossi, E., Norling, B., Persson, B., and Ernster, L. (1970) Eur. J. Biochem. 2, 508-513. Giuditta, A., and Singer, T.P. (1959) J. Biol. Chem. 234, 666-671. Schewe, T., Rapoport, S., Bbhme, G., and Kunz, W .p73) Acta. biol. med. Germ. 31, 73-86. Grossman, S., Goldenberg, J., Oestreicher, G., Kearney, E.B., and Singer, T.P. (1974) unpublished data. Georgopoulos, S .G., Chrysayi, M., and White, G .A. ( 1975) Pesticide Biochem. Physiol . 5, 543-551. Ackrell ,I .A.C . , Kearney , E .B., Mowery, P., Singer, T .P., Beinert, H., Vinogradov, A.D., and White, G .A. (1976) in Iron and Copper Proteins (Yasunobu, K., ed.), Plenum Press, New York, in press. L. (1971) Biochem. Biophys. Nelson, B .D . , Norling , B ., Persson, 9. , and Ernster, Res. Communs. 44, 1312-1320. -

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