Internal electron transfer within mitochondrial succinate-cytochrome C reductase

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

December 29,1978

Pages

INTERNAL MITOCHONDRIAL

1261-1267

ELECTRON TRANSFER WITHIN SUCCINATE-CYTOCHROME

C REDUCTASE

Yigal Ilan Department of Physical Chemistry Hebrew University, Jerusalem (Israel) Received

October

3, 1978

SUMMARY: Internal electron transfer within succinate-cytochrome C reductase from pigeon breast muscle mitochondria was followed by the pulse radiolytic technique. The electron equivalent is transferred from an unknown donor to b type cytochrome(s]I in a first order process with a rate constant of: 660+15Os . This process might be the rate determining step of electron transfer in mitochondria, since it is similar in rate to the turn over number of the mitochondrial respiratory chain. INTRODUCTION Succinate-cytochrome breast

mitochondria

chrome

b565,

contains

cytochrome

iron-sulfur

protein

of the

mitochondrial

tivity

includes

cytochrome energy

c reductase

b561,

(1).

It

from

the

to get

of electron

Its

reduced

processes

cytoRieske

complex

III

catalytic

ac-

coenzyme

electrochemical

into

c reductase,

Q to free

in ATP molecules the

catalytic

(2).

activity

we have

undertaken

the

between

components

of

complex. The technique

to

(2).

transfer

some insight

and the

called

from

electron

transfer

cl,

chain

of the

from pigeon

components:

frequently

transfer

of succinate-cytochrome

this

is

c, and conservation

derived

redox

cytochrome

respiratory electron

In order

study

several

isolated

reduce

reduction,

the

complex

an internal

of pulse in

radiolysis

a very

electron

short transfer

(3) was used, time.

After

process

the

in

order

primary

to cytochrome(s)

b was followed. 0006-291X/78/0854-1261$01.0~/0 1261

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

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

Succinate-cytochrome c reductase was isolated from pigeon muscle mitochondria by the procedure of Erecinska et al. as modified by Leigh and Erecinska (5). The properties and (41, constitution of this preparation were given by Erecinska et al. (1). HCOONa was supplied by B.D.H., Na2HPO4 and NaH2P04 by Mallinckrodt, t-butanol by Merck, and ethanol by Fluka. All reagents were analytical grade reagents. N20 and argon were supplied by Israel Oxygen Centre. The Varian linear accelerator of the Hebrew University and the optical and electronic systems were described elsewhere (6). The irradiation cell was a 1x1~2 cm rectangular flow-through cell fabricated from spectrosil. The optical path was 3 cm, by passing the analytical light three times through the cell. Spectra of solutions were taken on a Gary 14 spectrophotometer, and pH measurements were made with a digital pH meter (pHM52 Radiometer). The pH of the solutions was set by phosphate buffer (10m4 10'3M). Solutions were prepared in glass bottles 2 hours, or less, before irradiation. Deaeration of solutions and saturation with N20 were accomplished by sweeping with argon or N20 for at least 15 minutes, in large glass syringes equipped with capillary standard taper joints. Irradiation was carried out no more than 0.5 hours after sweeping with the gas. The concentration of succinate-cytochrome c reductase was determined by measuring the absorption difference between the dithionite reduced and ferricyanide oxidized protein, using the extinction coefficients as given by Wilson and Erecinska (7). The absorbed dose per pulse was determined routinely using the spectrum of hydrated electrons produced by pulsing 1x10e2M aqueous ethanol at pH = 9.5-10, taking cS7g = l.O6x104M-lcm-1, and G(ea ) = 2.75 molecules/lOOeV (8). Doses used ranged between 200 and 3500 rads per pulse (equivalent to 5x10-7-1x10-5M of of the solutions was 18*2"C. eiq) . The temperature The data were analyzed on-line using a Nova 1200 minicomputer and appropriate interphases developed in our laboratory. breast

RESULTS Succinate-cytochrome

c reductase

was reduced

by three

radi-

cals: a. e 0.05M

aq' t-butanol,

Solutions

5x10e4M

saturated

are scavenged electron b.

CO,.

contained

and 10m4 -10m3M

were

radicals by the

Solutions

pulse Solutions

phosphate

buffer

with

6x10m7phosphate

argon.

by t-butanol reduce

the

of

buffer

Under (3).

these

the

protein,

(pH = 7.2 - 7.5). conditions,

eaq and H'

atoms

OH' formed

protein.

contained (pH = 7.4)

1262

6x10s6M

(l-2)x10m6M and O.lM

of

the

HCOONa,

protein, and were

satu

BIOCHEMICAL

Vol. 85, No. 4, 1978

rated

with

N20.

the pulse

are

Under

these

transformed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

conditions, within

all

lpsec

the

into

radicals

CO,,

formed

by

by reactions

with

N20 and HCOO- (3). c. CH3CHOH.

Solutions

5x10m4M phosphate

buffer

turated

Under

with

N20.

contained (pH = 7.4) these

OH radicals

by reaction

mainly

CH3CHOH radicals

into

The results were

similar

after

increase

330-390

of

fast

absorption

changes

not

The general

drawn,

increase This

at

shape

complex. tion

all

When the

seconds proved

after it

660%150 the

ionic

concentrations experimental

fast

at the

agent

was eaq' was also

decay

conditions,

the

is

the

spectrum

was

wavelengths.

components

we followed too

The

of an absorption

shorter

of any of the

< ranges:

to be analyzed.

spectrum

higher

(~1,~

wavelength

and therefore

of this

(see below) used.

experimental

were too

(9).

a fast

in the

into

fast

of

its

the

absorp-

to be analyzed

set). change

the pulse,

could

to be a first s-1

radicals

sa-

transformed

ethanol

reducing

the

characteristic

This

are

were observed

small,

shape

transformed

that

was observed

were very

reducing

with

protein, and were

ethanol,

was completed,

nm. Under

an absorption

Later,

three

process

at 600 nm.

-t 0.5

('cl/2

the

of the

e- is aq OH' radicals

by a reaction

wavelengths,

is not

decay

N20 (3).

in absorption

this

conditions,

the pulse

nm, and 420-580

kinetics

and O.lM

processes

each of

Immediately 0.5~)

with

in both

for

2x10e6M

(Table

1).

strength,

of

process

The rate

of this

the

of

(Table

type the

was completed

be observed.

order

of either error

which

radicals

1).

1263

with

Its

reducing or the

kinetic

a rate

process

-5 millianalysis

constant

of

was independent

radical, protein,

of

and of the within

the

Vol. 85, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1 Rate constants of electron transfer to cytochrome b within succinate-cytochrome c reductase. The concentration of the radicals -7 (eao, COi,CH5CHOH) were: 5x10 - 1x10m5M. pH = 7.2-7.5. The kinetics were followed at the wavelengths ranges: 425-450 nm and 555-565 nm. reducing

concentration the protein

radical

eaq

co,

CH3CHOH

of P

(lo-6M)

1

0.002

2

0.004

(3)

55Ok

3

0.006

(5)

5

0.01

(4)

750+100 810+150

0.6

0.1

(4)

510+ 70

2

0.005

(4)

700+100

(a) In brackets - the number of independent The mean value: 660?150 s-1

The difference pulse trum

is

shown in

between

spectrum figure

reduced

dicals not

caused probably

transfer In

(550 nm, tion decay that

It

the

reduction

of

with

an electron

similar

Only

immediately

we could

to the reduced

the

reduction in

the

to cytochrome

1264

difference

5% -10% of

b,

after

spec-

the

reducing

b.

The rest

of

the

complex

which

or with

the

cytochrome

observe

the

(r

l/2

of cytochrome fast

process b.

ra-

do

impurities.

and oxidized

pulse

the

b (4).

cytochrome

of reduced

after

milliseconds to the

of

80

experiments.

cytochrome

components

438 nm and 420 nm),

equivalent

ten

to cytochrome

points

substance

is

was low:

reacted

in parallel

an electron

yield

isosbestic

formed

the

1.

and oxidized

The reduction radicals

obtained

(14) 620*150

that

< 0.5usec), b.

This

does not

fast

b absorp

did

not

indicates transfer

BIOCHEMICAL

Vol. 85, No. 4, 1978

Figure

1:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Difference spectrum: reduced succinate-cytochrome c reductase minus the oxidized protein, ~10 milliseconds after the electron pulse. The reducing agent eaq. Concentration of the protein: 3x10-6M. pH=7.4. The dotted line - difference spectrum of cytochrome b (dithionite reduced succinate cytochrome c reductase? minus the ascorbate reduced protein), after Ereclnska et al. [4].

DISCUSSION The slow process

within

by the

facts

of

radical

the

ionic

reduction

that

its

type

strength,

1),

kinetics

is

similar

first

concentrations

of the

difference

c reductase

contains

enough

- b 565 ana b561The experimental to allow a determination whether

one of

these

b cytochromes,

or are both

1265

after

is

supported

radicals),

the

reactants. this

internal

spectrum

conditions spectrum

reduced

process

of cytochrome

two types

this

an internal

and are independent

and uncharged

obtained

to the

1) is This

order,

charged

spectrum

(Table

c reductase. are

(including

or the

Succinate-cytochrome chromes

observed

succinate-cytochrome

The difference (figure

process

b.

of b cytowere not belongs

at a similar

good to rate.

BIOCHEMICAL

Vol. 85, No. 4, 1978

The rate

constant

is similar

s-3, piratory

to the

chain

These

to

b.

In previous or the

in

situ

what

with

therefore

could

the

rate

between

the

its

could

rate

of

electron

transfer

of the

mitochondrial

process transfer

either

(660+150 res-

under

steady-state

the in

studv

an electron

an electron

reactions

such that

this

internal

rate

was a fast

step and the

piratory

chain.

The results

internal

source

of

in vivo,

obtained

the electron

This

number until

equivalent

were

step

study. of rea-

an accelerator, did

to cytochrome latter

of the

step by the

could

be

similarity res-

reveal

reduces

not b, which

mitochondrial

now do not which

fol-

conditions.

c reductase

as is evident

turnover

equivalent

generation

from

transfer

separately.

remain

determining

the present

pulse

electron

< 0.5~)

slower

be followed in

(r1,2

of succinate-cytochrome

be followed

determining

fast

do not

were

used

reduction

the

was investigated

by means

interfere

number

(Z),

systems

than

The primary

species

system

The technique gents

in

studies

The experimental was slower

internal

turnover

formed

unidentified.

lowed,

the

RESEARCH COMMUNICATIONS

(10).

The species

cytochrome

of

AND BIOPHYSICAL

the

cytochrome

b. ACKNOWLEDGEMENT: The very willing help of Dr. M. Erecinska with the preparation of succinate-cytochrome c reductase is acknowledged. for valuable discusI thank Prof. G. Czapski and Dr. A. Shafferman sions. I especially acknowledge the help and incentive of the late Prof. G. Stein, at the beginning of this work. This research was supported by ERDA under contracts E(ll-1)3009 and E(ll-1)3221. REFERENCES ]lJ [Z] [3]

Erecinska, M., Wilson, D.F. and Miyata, Y. (1976) Arch. Biothem. Biophys. 177, 133-143. Rieske, J.S. (1976) Biochim. Biophys. Acta 456, 195-247. Shafferman, A. and Stein, G. (1975) Biochim. Biophys. Acta, Bioenerg. Rev, 2, 287-317.

1266

Vol. 85, No. 4, 1978

r41 [51

161 [71

[81 191

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Erecinska, M., Oshino, R., Oshino, N. and Chance, B. (1973) Arch. Biochem. Biophys. 157, 431-445. Leigh, J.S. Jr. and Erecinska, M. (1975) Biochim. Biophys. Acta 387, 409-421. Lichtin, N.N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117-135. Wilson, D.F. and Erecinska, M. (1975) Arch. Biochem. Biophys. 167, 116-128. Hart, E.J. and Filden, E.M. (1970) in: "Manual of Radiation Dosimetry" (Holm, N.W. and Berry, R.J., eds.) pp. 331-335, Marcel Dekker Inc. New York. Dorfman, L.M. and Adams, G.E. (1973) Reactivity of the Hydroxyl Radical in Aqueous Solutions. NBS report No. NSRDS NBS 43.

[lOI

Chance, B. (1969) (Morios, M., ed.) Amsterdam.

Physics in: "Theoretical pp'. 156-158 North-Holland

1267

and Biology" Publishing

Co.