Ubisemiquinone radicals from the cytochrome b−c1 complex of the mitochondrial electron transport chain—Demonstration of QP-S radical formation

Vol. 99, No. 4,198l April

BIOCHEMICAL

AND EIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1411-1419

30, 1981

UBISEMIQUINONE RADICALS FROM THE CYTOCHROME b-c1 COMPLEX OF THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN--DEMONSTRATION OF QP-S'WICAL FORMATION Yau-huei Wei, Charles P. Scholes and Tsoo E. King Departments of Chemistry and Physics State University of New York at Albany, Albany, New York 12222

Received

March

16,

1981 Summary

Stable ubisemiquinone radical(s) in the cytochrome b-c -II complex of 1n the presence of bovine heart was observed following reduction by succinaTecatalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Qa. The radical showed an EPR signal of g = 2.0046 + .003 at X band ("9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 + -5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved g--anisotropy and had a field separation between derivative extrema This radical is evidently from QP-C. These observations of 26 f 1 Gauss. substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome complex only in the --b-cl-II presence of more than a catalytic amount of succinate dehydrogenase and cytochrome --b-c . This signal was not antimycin A inhibitory. The signal amplienzymic activity of succinate-cytochrome c tude paral 1 eled the reconstitutive reductase from succinate dehydrogenase and the cytochrome complex. -b-cl-11 -Introduction: made more than none

in

Although twenty

the discovery

years

the mitochondrial

ago

(1,2),

electron

the

of ubiquinone sequence

transport

chain

in mitochondria

and mechanism 1

is

still

not

was

of ubiquiclear

(see

n

e.g.

3,4).

nate-Q

The recent

reductase

reconstitution dehydrogenase

discovery

segment of the

of a Q-binding

of the respiratory

so-called

succinate-Q

(SDH) and QP-S (8,9)

1 Electron transfer terms) used is actually or all other species if transfer.

has cast

protein

chain

(5-7)

reductase certain

(QP-S)‘

in

the succi-

and the successful from

doubt

soluble about

succinate the mobile

(electron transport, electron pathway, or related an abbreviation including the transport of hydrogen those species indeed occur in the respiratory chain

n

'DPPH, a, or-diphenyl-S-picrylhydrozyl; Q, ubiquinone, the subscript indicates the number of isoprenoid units; QP, ubiquinone bound to a specific protein; QP-C, ubiquinone protein that occurs in the cytochrome --b-c l region; protein that exists in the NADH dehydrogenase segment; QP-N, a ubiquinone QP-S, a ubiquinone protein that accepts electrons directly from SDH; QHa fully reduced ubiquinone; SDH, soluble, reconstitutively active succinate dehydrogenase: TTFA, theonyltrifluoroacetone.

Vol. 99, No. 4,198l

nature

as well

obtained

BIOCHEMICAL

as Q-pool

thus

far

AND

theory

has indicated

BIOPHYSICAL

of ubiquinone

(10-12).

the

of at least

existence

binding

proteins

in

the respiratory

chain

quinone

radicals

in

the cytochrome

--b-cl -III3

genase

segment

(QP-N)

have been

in

the order

Trumpower

(15)

radicals

exist

to demonstrate have

nase

only

have in

favor

claimed

(3,13).

form

two different

--b-cl.

of QP-S radicals

(cf -*

that

the

In this

succinate-Q QP-S radical(s)

communication

and other

EPR properties

in bovine

heart

ubisemi-

2

constant Ohnishi

is and

of ubisemiquinone

However,

active

Q-

and NADH dehydro-

3,4).

populations

of reconstitutively

radical(s)

(QP-C)

-c reductase.

found

stable

The dismutation

QP-S in the reconstituted we have

evidence

two additional

In fact,

(3,14).

COMMUNICATIONS

Collective

complex

of a non-radical that

in the presence

on the ubisemiquinone

reported

succinate-cytochrome

isolated

and cytochrome

stration

in

Recently

failed.

strated

of lOlo

RESEARCH

numerous reductase

including cytochrome

system

may be demon-

succinate

we wish

efforts

to report Q-band --b-cl-II

dehydrogethe

demon-

spectra complex.

Materials and Methods: The preparations of SDH and the cytochrome --b-clII complex were made and assayed essentially as previously reported (16) with some modifications. X-band EPR measurements were conducted in a Varian E-4 EPR spectrometer and Bruker ER-420 EPR spectrometer at room and liquid nitrogen temperatures. Q-band EPR studies at 35.0 GHz were done in a Varian nitrogen temperatures. Model V-450214503 spectrometer at room, ice and liquid Fumarate and TTFA were recrystallized from ethanol. Qz was synthesized in this laboratory (14). Since the ubisemiquinone radical was found to be rather sensitive to ethanol, both TTFA and antimycin A were dissolved in dimethylsulfoxide and stored at 0-4°C. A flat EPR cuvette was used for room temperature study in a Varian E-4 EPR spectrometer. About 0.4 mm diameter capillaries The ice temperature was obtained were employed for the Q-band EPR studies, by putting the Q-band cavity into a small Dewar containing an ice-water mixture. A small flat cell that fits into the Bruker B-VT 1000 variable temperature flow Dewar was used for controlled temperature X-band work. At X-band a Hewlett Packard Model 52408 12.4 GHz Frequency Counter and a Harvey-Wells Model G502 NMR probe were used for measuring the EPR frequency and magnetic field strengths needed to compute g-values. At Q band a very small piece of DPPH (with known g-value of 2.0036) was placed in the EPR cavity for some experiments, and g-values of the ubiquinone radical were measured with respect to it. The magnetic field sweep range used at Q band had been calibrated by 3 The cytochrome complex is prepared from succinate-cytochrome c --b-cl-1 reductase and is insoluble. The cytochrome complex is identical --b-cl-II functionally with the b-cl-I complex except its aqueous solubility. Both can -reconstitute with soluble SDH to form succinate-cytochrome c reductase. Removal of QP-S from the -b-cl-II complex yields the cytochrome com--b-cl-III plex and thus the complex is reconstitutively inactive with SDH. However, succinate-cytochrome 2 reductase is physically and functionally formed by admixture of the --b-cl-111 complex, QP-S and SDH (8).

1412

3

BIOCHEMICAL

Vol. 99, No. 4,198l

OO L/,

AND

BIOPHYSICAL

4/

6

12 1

1

RESEARCH

J

COMMUNICATIONS

16 II

TIME (mini

Fig.

Correlation of the reductions of cytochrome band 21 with the formation of protein-bound ubisemiquinone radical in cytochrome b-cl-II complex. The system used for a and ~1 reduction contained i.2 mg b-cl-11 complex (9.1 nmolb and 4.8 nmol 21) 7.5 x low3 nmol SDH and i.7 nmol Qz in 0.72 ml total volume. The reductions of b and c were followed right after addition of 1.0 ~1 of a substrate mix-t ure, containing 60 mM fumarate and 15 mM succinate, by following the difference spectra in a Amico DW-2 spectrophotometer using an identical sample without substrate as reference. The system used for Q radical signal measurement in a Varian E-4 EPR spectrometer contained 10.8 mg b-cl-II complex (82 nmolb and 43 nmolcl), 6.7 x lo-* nmol SDH and i7-nmol Q2 in 0.18 ml total volume. The Q radical production was initiated by addition of a 10 nl substrate mixture containing 60 mM fumarate and 15 mE1succinate to the system and the signal was monitored at the peak of absorption with the machine settings of microwave frequency 9.492 GHz, microwave power 100 mW, 100 KHz field modulation of 5.0 Gauss amplitude, time constant 1.0 set, scan rate 50 Gauss/min. The EPR and optical recordings are all carried out at 23°C.

1.

reference to the 32 Gauss splitting of the outer features from a mobile nitroxide spin label. Signal to noise of the Q band work and X-band Bruker work was enhanced by repetitive spectral accumulation in a Tracer 570 Signal Averager. Results study

and Discussions:

contained

not

less

2.0 nmol ubiquinone in electron be initiated amounts dependent Before

tion

This

radical

b (cf.

this

segment

reduced

to the degree

Fig.

cytochrome

was stable

appeared

--b-cl -11

radical(s)

oxygen,

of reduction of cytochrome

plus

The ubisemiquinone

chain

radical

could

catalytic

of cytochromes

concomitantly

b,

involved

and showed

~1 almost

in this

cytochrome

of the respiratory

towards

1413

used

6.3 nmol

Qs or succinate

approximately 1).

~1,

complex

The ubisemiquinone

of the reduction

and the signal

of cytochrome

in

of either

related

the completion

observed,

3.5 nmol

reactions

by addition

change,

than

per mg protein.

transfer

of SDH.

The cytochrome

a timec -b and -1'

no signal with

was

the reduc-

was found

to

BIOCHEMICAL

Vol. 99, No. 4,1981

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

THE k-c,-IICOYPLEX 50

Fig.

2.

be greatly

The effect of the fumarate to succinate ratio on the stability of the protein-bound ubisemiquinone radical. The systems all contained 12.6 mg b-c -11 complex (75 nmol b and 44 nmol c ), 0.12 nmol SDH and 185 Km: 1 Qs in a total volume-of 0.22 ml. Ekfferent ratios of fumarate-succinate mixture were added to the reconstituted enzyme solution and the RPR signals of the Q radical formed thereof were monitored under identical Varian E-4 machine settings of microwave frequency 9.492 GRz, microwave power 100 mW, 100 KHz field modulation of 8.2 Gauss amplitude, time constant 3.0 see, scan rate 25 Gauss/min. The signals were followed until equilibrium was reached. The total concentration of fumarate plus succinate was maintained at about 25 mM in each system.

stabilized

and intensity Fig.

2.

ratio the

were

dependent

The maximal

approached radical

4.

fumarate

to succinate

rapidly,

reached

various

In the

range

slowly

fumarate

were

and then

by titration to succinate This

for

oxidized

QP-C reduced/QP-C couple)

using

The ubisemiquinone of catalytic g=

2.0046

amount + .0003

is

at s9.4

GHz with

several

of 5 to 20, hours

When mixtures

the ubisemiquinone

radical

to a constant amplitude

protein -b-cl-III --

in cytochrome

SDH showed

for

an EPR signal no resolved

1414

bound

even at of lower

formed

level

(cf.,

of ubisemiquinone

with

hyperfine

of Em

bound

(17).

complex

of typical

against

value

ubiquinol/protein

--b-cl-II

Fig.

+50 mV at 23" and

the overall

complex

in

to succinate

was reached.

in agreement

the cytochrome

as shown

ratios

a Em of approximately

(i.e.

radical of

constant

of signal

value

ratio,

to succinate

decreased

ratios,

pH 7.4 was obtained.

ubiquinone

used,

appearance

when the fumarate

of fumarate

height

signal

to succinate

and remained

ratios

and its

fumarate,

was reached

the maximal

a maximum,

At equilibrium,

2) *

height

after

of

on the fumarate

signal

was formed

room temperature

the presence

in

free

in the presence radical

structure.

at The line

BIOCHEMICAL

Vol. 99, No. 4,198l

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MAGNETIC FIELD (GAUSS1

Fig.

The EPR spectrum of the protein-bound ubisemiquinone radical in the cytochrome b-c,-11 complex observed in Q-band RPR spectrometer. The system contai;ed about 0.01 ml sample in a thin capillary EPR tube (0.04 x 8 cm). The sample contained 0.48 mg b-c -II complex (9.7 nmol -1c with b_- to --1 c ratio of about 1.8), 2 x-15" nmol SDH, 8 nmol Q2 and 120 nmol fumarate and 45 nmol succinate as substrate in a total volume of 0.01 ml. The spectrum was taken at ice (%O"C) temperature on a Varian Model V-4502/4503 Q-band EPR spectrometer using the settings of microwave frequency 34.99 GHz, microwave power

3.

10 dB (%lO mW), 100 Kllz field modulation of 4 Gauss amplitude, constant of 0.1 set, and scan rate 100 Gauss/min, with a total 3 accumulations in the signal averager.

width

of this

signal,

estimated

at room temperature

and

the ubisemiquinone,

i.e.

8.4

f

anisotropic

It

a field

showed

illustrated ble

strongly

reaction

those

centers

the QP-C radical none radical 4

The ice disappearance

at

EPR spectrum

3 taken

at ice

reported

at

strongly not

1.3"K

(sO"C)

--et al.

(18).

a freely

and would

temperature and room temperature of the radical signal was slower

amounts

+

0.5

when

of a and 77°K.

of 26 ? 1 Gauss, These

in bacterial

since

conditions,

at room temperature

temperature.4

by Feher

these

8.1

EPR spectrometer,

extrema

radical

was

suitable

in a Q-band

derivative

immobilized

showg-anisotropy,

with

was observed

between

extrema,

Under

77°K.

formed

of ubisemiquinone

is

would

Gauss

was examined

separation

in Fig.

0.5

the derivative

QP-C radical,

fumarate-succinate-mixture, prominent

between

time of

spectra

as resem-

photosynthetic This tumbling

show resolved

indicates

that

ubisemiquihyperfine

EPR spectra were identical at ice temperature.

but

Vol. 99, No. 4,198l

structure, These

BIOCHEMICAL

and substantial

observations

immobilized

line

clearly

and bound

of excess

remained

(21);

On the other reason

quinone this

hand, for

could

signal

above was found

~1 in

the

inhibition

of this

radical

is not

amount

clear

completely

of SDH, was the in the

cytochrome

although

sufficient

amount

of QP-S was present

simple

ubisemiquinone When the amount sensitive

as well

as at 77".

added

the enzymic lation

complex

activity

indicated.

It

with

less

amplitude

most

likely

Q (22,23).

It

is

concluded in

appeared under

the

complete;

this

complex

systems,

ubisemi-

the presence no QP-S

conditions

system

to generate

another

EPR signal

was found

to be approximately

signal

11 Gauss at

was proportional The signal

systems.

to the

amplitude

Figure

4 shows the

of SDH to the cytochrome

might

be noted

that

the

SDH was used;

this

semiquinone comparable

radical

width

of SDH which

center

1416

corre-

with

of this

was evidently

to the ubisemiquinone. interaction

e.g.

line

activity

SDH

paralleled

EPR signal

at ratios

which

at room temperature

2 reductase

species,

that

that

the

at g = 2.00

the systems.

due to the spin-spin

paramagnetic

with

succinate-cytochrome

the flavin intensity

in

A appeared

of this

in

when more and more

overlapping

radical

Since

A, it

complex

the

of the reconstituted

A-insensitive

increased

another

complex

of reconstituted

antimycin

width

The amplitude

to the --b-cl-II

in

antimycin

The line

room temperature.

in the

of QP-S.

of SDH increased

towards

antibiotic

However,

competes

b-cl-11 --

--b-cl -11

to

ubisemiquinone

QP-C radical.

was involved

radical

system.

at present.

cytochrome

sensitive

by TTFA was never

by antimycin

from

is

of this

Q 2, some residual antimycin

was formed

to be very

the

(19).

(20).

by addition

because

which

was not

radical

apparently

incompleteness

of a catalytic

ubisemiquinone earlier

abolished

COMMUNICATIONS

of temperature

as proposed

added

RESEARCH

upon change

is

be abolished

radical,

this

to cytochrome

exogenously

this

changes

that

be completely

concentration

presence

the

indicate

described

A and could

at equimolar

shape

to a protein

The ubisemiquinone antimycin

AND BIOPHYSICAL

the b-c -- l-II

signal

was

due to spectral possesses The signal

of ubisemiquinone S-3 of SDH as suggested

much is

of QP-S with by Beinert

BIOCHEMICAL

Vol. 99, No. 4,198l

AND

BIOPHYSICAL

WHIP-&-II

Fig.

4.

(personal --b-cl

communication).

abolished

flux

that

to the steady

involving

this

signal

substantiated

QP-S is is

-b-cl-_ -11

SDH was present

of Qe.

state being

kinetics further

radical

investigated.

plus

amounts

a catalytic (e.g.

of the ubisemiquinone The half-time

that

generated

1417

antimycin

by addition amount

reduction These

This

facts

conclusion

A completely of exogenous

of SDH, but

not

QZHZ so when

>20%). radicals

to reach

of the cytochrome

of cytochrome

due to the QP-S ubisemiquinone.

complex

in greater

The formation

of the presence

by the observation

the ubisemiquinone

to cytochrome

by addition

mole ratio

The requirement

related

and electron

was further

COMMUNICATIONS

Correlation of reconstituted succinate-cytochrome c activity and the antimycin A-insensitive EPR signal amplitude. Reconstituted reductase preparation was made from different amounts of SDH to the b-cl-11 complex as shown by the mole ratios on abscissa. The enzymic activity (*) of each system was assayed spectrophotometrically by following the reduction of cytochrome 2 at 550 nm. The reconstituted reductase preparation for EPR studies were identical with those used for activity assay and contained 20 to 70 mg per ml solution. For RPR studies, each system was preincubated with 4 ~1 of 10 mM antimycin A for 10 minutes at 4°C. The EPR signals were initiated by addition of 15 ~1 substrate solution containing 60 mM fumarate and 10 mM succinate, and the spectra were recorded after the signals reached equilibrium at 23°C. The signal amplitude (A) was normalized on the basis of cytochrome --1 c concentration in each system.

apparently

indicate

RESEARCH

was found

the maximal

signal

to be stimulated height

was

Vol. 99, No. 4,198l

decreased

when

BIOCHEMICAL

the concentration

of the ubisemiquinone

complex

of this

only

Q2 titration This

a fraction that

deficiency

transfer

activity

required

more exogenous

An optimum complex

level

was found

again

of that

of ubiquinone and both

of the

in this

antimycin

Q than

which

A sensitive

for

the antimycin

summation

of exogenous

to be two mol of Q per

supports

the existence

Acknowledgement--Experimental from NIH and the American Cancer

cl,

40% --b-cl-

the results

was deficient

in

of the preparation

is known

segment

about

of the cytochrome

complex

a result

intensity

to increase

of cytochrome

the b-cl-11 --

fractionation

COMMUNICATIONS

The EPR signal

content

was apparently

ammonium sulfate-cholate The amount

RESEARCH

was found

the ubiquinone

indicates

Q (1’3).

tion

at equilibrium

Since

was usually

ubiquinone. ing

Q2.

EIOPHYSICAL

of Q2 increased.

radical

by externally-added II

AND

for

to be able

showing

to remove

maximal

and insensitive

electron

EPR signals

A-sensitive

signal

and endogenous

mol of cytochrome

involv-

alone.

Q in the --b-cl The observa-

cl.

of QP-S and QP-C.

work was generously Society.

supported

by grants

References 1.

Green, D. E. (1961) in 'Quinones Wolstenholme and C.M. O'Connor,

2.

Morton, London.

3.

King, T. E. (1980) in to Professor K. Yagi" eds.), Japan Scientific

4.

King, Vol.

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Yu, C. A., Yu, L. and King, 78, 259-265.

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King, T. E., Yu, L., Nagaoka, S., Widger, W. R., and Yu, C. A. (1978) in "Frontiers of Biological Energetics" Vol. 1 (L. Duttin, J. Leigh, and T. Scarpa, eds.) Academic Press, New York, pp. 174-182.

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(in