Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

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A NEWMEMBRANE IRON-SULFURFLAVOPROTEINOF THE MITOCHONDRIAL ELECTRONTRANSFERSYSTEM THE ENTRANCEPOINT OF THE FAll'Y ACYL DEHYDROGENATION PATHWAY?* Frank J. Ruzicka and Helmut Beinert Institute for Enzyme Research University of Wisconsin Madison, Wisconsin 53706 Received

July

18,1975

SUMMARY: An iron-sulfur (Fe-S) protein has been purified from beef heart mitochondria by following its EPR signal after reduction, which is characteristic of a ferredoxin-type Fe-S protein (g, = 1.886; gy = 1.939; g, = 2.086). The signal intensity corresponds to one unpaired spin for 4 to 5 Fe atoms. The light absorption spectrum indicates the presence of flavin. Fe, labile S, and FAD are released by acid at a ratio of approximately 4:4:1. Neither prosthetic group of the protein is reduced by NADH, NADPH, succinate, glycerol-3-phosphate or dihydroorotate. The Fe-S group is, however, reduced with a half-time of QJ~msec, when the protein is mixed with an equivalent amount of electron transferring flavoprotein (ETF) of the &oxidation cycle, prereduced with an acyl CoA dehydrogenase and a saturated fatty acyl CoA. In the presence of the two added flavoproteins the behavior of the flavin of the Fe-S flavoprotein could not be determined. Complexes I-III are not reduced by reduced ETF under analogous conditions. The low field EPR resonance ["center 5", Ohnishi et al. (1972), Biochem. Biophys. Res. Commun. 46, 1631-16381 of the protein is read?iyTbserved in whole tissue, mitochondria and sonic fragments from all species we have examined. Therefore, the protein appears to be a universal constituent of mitochondrial electron transfer systems. of the resonances observed in heart

Most

at presently

attainable

are yet unidentified (width

at half

height

of a ferredoxin-type parallel "center

have been identified.

Among those that

in the reduced state

Q20G) which has properties signal.

5" by Ohnishi -et al. soluble

by EPR spectroscopy

is a resonance present

(1).

of this

at g = 2.086 component (g,) resonance go

These resonances have been attributed The signal

preparations

to the observed signal

of a low field

Changes in the intensity

with changes at g = 1.89.

or in purified cording

sensitivities

mitochondria

of NADHor succinate

intensity,

ing component is of the order of that

is not present

of the individual

in Complex I-III

dehydrogenase.

the concentration

to a

Ac-

of the correspond-

Fe-S centers

of NADH

*This work was supported by a research grant (GM-12394), a research career award (S-K06-GM-18,442) to H. B. and a special fellowship (5F03GM55117) to F. J. R. by of Health. the Institute of General Medical Sciences, National Institutes

Vol. 66, No. 2, 1975

AND BIOPHYSICAL

We have now been able to purify

dehydrogenase. by cholate

BIOCHEMICAL

ammonium sulfate

solubilization,

chromatography,

a protein

from mitochondria

fractionation

and DEAE-sepharose

in which these resonances can be observed after

dithionite.

The complete EPR spectrum of this

Fe-S protein

containing

such as, e.g.,

RESEARCH COMMUNICATIONS

a component which can exist

flavin

but no detectable

protein

or ubiquinone

(Q).

reduction

is typical

of that

in a free radical

The protein

with of a

state,

was found to contain

FAD

Q.

MATERIALSAND METHODS. Beef heart purification

mitochondria

procedures

acyl CoA dehydrogenase purified

were carried

and the electron

manufactured

Dr. T. P. Singer.

transferring

and flavin

ashing after

et al. --

(9).

Q was determined

phoresis

separating

by sulfuric

Wisconsin.

to Fairbanks

et al.

S (7)

acid.

Chromatography

of flavins

out according after

to Kilgour

extraction

(10).

to Davis (11) utilizing

5%

X100. (12).

dehydrogenases were performed

labile

Fe was also determined

out according

of

of Sigma Chemical Company.

work.

spectrophotometrically

gels with 0.5% Triton

was according

phate and choline

as in previous

was carried

fatty

(3,4).

(5) and Fe (6),

at 80' for 20 min was carried

Disc electrophoresis acrylamide

out as described

denaturation

deproteinization

All

(ETF) were

procedures

Milwaukee,

acid was a product

was carried

(8) were determined

(general)

flavoprotein

of described

(2).

by Eastern Chemical Company, was a gift

L-Dihydroorotic

EPR spectroscopy

to Crane et --* al The yellow

CoA was purchased from P-L Laboratories,

Glycerol-3-phosphate,

without

according

out at O-4'.

from beef heart by a modification

n-Butyryl

after

were prepared

SDS-polyacrylamide

electro-

Assays for glycerol-3-phosaccording

to Ref.

(13).

RESULTS Purification 0.25 -M sucrose,

of Fe-S flavoprotein. 0.01 M - Tris-HCl

and 1 mM - succinate, of 30 mg per ml.

Mitochondria

(pH 7.4) containing

and suspended in the same buffer Potassium cholate

were washed once in 1 mf$ dithiothreitol to a protein

concentration

(20% w/v, pH 7.6) was added slowly

623

(DTT)

to a

Vol. 66, No. 2, 1975

concentration

BIOCHEMICAL

of 0.25 mg per mg of protein.

20 min and centrifuged a protein

sium succinate

was added to a concentration

tion

was stirred

for 2 hrs.

stirred

of solid

column equilibrated

with the same buffer

column application,

the protein

40,000 x g for 30 min.

fate

at pH 7.4.

contained

(pH 7.4),

(5 cm) DEAE-sepharose

as used for dialysis.

The fraction

was fractionated

precipitated

the Fe-S protein.

Polyacrylamide

fragments

A preparation

it

from the other preparations,

to be approximately consists

only of this

are taken into account,

ammonium

obtained

procedure

and Fe-S gave values practically is likely

The molecular

from the Fe-S flavoprotein.

estimated

at

are obtained

I, Table I) showed the same main component but different

obtained

protein

and centrifuged

in the presence of SDS or Triton

by a somewhat different

for flavin

between

paste.

sonicated

rived

eluted

15 mg protein

minor bands.

Since analyses

and a step-

with solid

showed one major band and several

ration

Following

between 45 and 55% ammonium sul-

Approximately

gel electrophoresis

mitochondrial

precipi-

for 3 hrs in 0.01 E Tris-HCl

1 mM_DTTwas collected

The supernatant

The fraction

from 200 ml mitochondrial

was fractionated

The fraction

to a short

was prepared.

The solu-

at 78,000 x g

was washed with the above buffer

(pH 7.4) gradient

of 1 M -

solution.

the Fe-S protein

dialyzed

1 mJJDTT, and applied

Then potassium

by the addition

ammonium sulfate.

(pH 7.4) containing

0.1 E and 0.25 MTris-HCl

Solid potas-

by centrifugation

which contained

tated between 50 and 60% was collected,

sulfate

followed

on ice for 20 min followed

at pH 7.4 by the addition

was resuspended to

on ice for 30 min.

(pH 7.4) to 0.25 ml per ml of protein

The supernatant

wise Tris-HCl

The pellet

on ice for

of 20 mM_,the pH of the solution

was added to 0.25 mg per mg protein

potassium phosphate

was stirred

of 30 mg per ml with the above buffer.

to 7.4, and the solution

cholate

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The mixture

for 3 hrs at 78,000 x g.

concentration

adjusted

AND BIOPHYSICAL

70,000. subunit

the protein

If it

that

624

identical

(prepa-

to those

the major band is de-

is assumed that

at the present

from

minor bands.

weight of this

and the results

Xl00

subunit was

the Fe-S flavo-

of the chemical analyses

stage is 25-30% pure.

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

625

RESEARCH COMMUNICATIONS

Vol. 66, No. 2, 1975

BIOCHEMICAL

EPR spectrum. ionite,

Fig.

of the purified

integration indicates

1 shows the EPR spectrum, protein

of the signal,

after

Varian rectangular

in at s 25 pwatt.

correction

cavity

The radical

signal,

after

reduction

with dith-

g = 1.939; gz = 2.086. Double Y for the superimposed radical signal,

spin for 4-5 Fe atoms (Table I).

that

of the signal

In the sets

in the presence of an excess of dith-

in the free radical

integrated

for the presence of approximately

RESEARCH COMMUNICATIONS

and at 13“K, saturation

It is interesting

the component represented

duced.

(gx = 1.886;

the presence of one unpaired

standard

ionite

AND BIOPHYSICAL

signal

is not further

re-

at 117'K and 90 pW of power, accounts

one unpaired

spin for every 3 molecules

of

flavin. Light

absorption

380 nm and a shoulder of a Fe-S flavoprotein. in all

preparations

(reduced).

spectrum. at 440-480,

The absorption

spectrum with a maximum at

which disappears

Contamination

by heme to a variable

from sharp absorption

However, in terms of iron,

on reduction,

this

lines

is suggestive

extent

at 417 nm (oxidized)

contamination

was evident and 425 nm

amounts to