Low temperature electron paramagnetic resonance studies on iron-sulfur centers in cardiac NADH dehydrogenase

Vol. 56, No. 3, 1974

BIOCHEMICAL

AND BlOPHYSlCAi

RESEARCH COMMUNICATIONS

LOWTEMPERATURE ELECTRONPARAMAGNETIC RESONANCE STUDIES ON IRON-SULFURCENTERSIN CARDIACNADH DEHYDROGENASE Tomoko Ohnishi Department

and John S. Leigh

of Biophysics and Physical Biochemistry Johnson Research Foundation University of Pennsylvania Philadelphia, Pennsylvania 19174 C. Ian Ragan* and E. Racker Section of Biochemistry and Molecular Biology Cornell University Ithaca, New York 14850

Received

December

5,1973

SUMMARY: EPR signals arising from at least seven iron-sulfur centers were resolved in both reconstitutively active and inactive NADtI dehydrogenases, as well as in particulate NADH-UQreductase (Complex I). EPR lineshapes of individual iron-sulfur centers in the active dehydrogenase are almost unchanged from that in Complex I. Iron-sulfur centers in the inactive dehydrogenase give broadened EPR spectra, suggesting that modification of iron-sulfur active centers is associated with loss of the reconstitutive activity of the dehydrogenase. With the reconstitutively active dehydrogenase, the E,B o value of Center N-2 (ironsulfur centers associated with NADH dehydrogenase are designated with prefix N) was shifted to a more negative value than in Complex I and restored to the original value on reconstitution of the enzyme with purified phospholipids. Ragan and Racker (1) have recently

reported

genase from NADH-UQreductase

[Complex I (2)],

with K3Fe(CN)o as an electron

acceptor,

Recombination

reductase

activity.

sensitive

NADH-UQreductase

Complex III (3).

This reconstitutively

the extraction ionite

to reconstitute

antimycin

NADH-UQ rotenone

combines with

NADH-cytochrome 5 reductase

is performed

775

activity

restores

preparation

NADHdehydrogenase

If these reducing

@ 19 74 tlv Academic Press. Inc. of reproduction in an,, form reserved.

full

phospholipids

A-sensitive

* Present address: Department of Chemistry, at Albany; Albany, New York 12222.

Cop.vright All rights

of NADH dehydro-

but lacks rotenone-sensitive

The resulting

of the enzyme by cholate

and dithiothreitol.

which retains

with purified

activity.

active

preparation

reagents

is obtained

only when

in the presence of dithare omitted

State University

during

of New York

the

Vol.

56,

No.

3, 1974

extraction, tive

BIOCHEMICAL

the resulting

UQ reductase

and inactive

AND

dehydrogenase

BIOPHYSICAL

contain

of non-heme iron and acid labile

sulfide,

not reveal

Recently,

seven (4)]

the respiratory particles

approximately

(< 77'K)

using mitochondria, In the present

centers

NADHdehydrogenases

EPR spectrometry

does

of multiple

in the NADH dehydrogenase region

or Complex I (7,8).

purified

analysis

the EPR characteristics

centers

iron-sulfur

active

the same concentrations

and spectrophotometric

chain have been studied

of individual

and inactive erature

iron-sulfur

(SMP) (5,6)

acteristics

(1).

of rotenone-sensi-

Reconstitutively

when combined with phospholipids.

any differences

COMMUNICATIONS

gives very low rates

NADHdehydrogenases

[at least

RESEARCH

submitochondrial investigation,

in both reconstitutively

were investigated,

and the redox titration

of

charactive

using low temptechnique

of Dutton

(9).

MATERIALSAND METHODS Reconstitutively

active

and inactive

from Complex I, as reported

by Ragan and Racker (1).

hydrogenase with phospholipids were kept frozen in liquid half-reduction according

to Dutton

as previously

was performed

nitrogen

potentials

NADH dehydrogenases were purified Reconstitution

as described

and thawed before

reported

(10).

previously

(1).

the experiments.

at pH 8.0 (EmgeO) were determined

(9) and Wilson -et al.

of the deSamples The

potentiometrically,

EPR measurements were performed

(11). RESULTSAND DISCUSSION

In the present dehydrogenase In a similar

communication,

are designated fashion,

genase were previously arising

from at least

Center N-la plus N-lb plus

N-6

(Fig.

and inactive

with prefix

iron-sulfur designated 7 iron-sulfur (Fig.

B)

centers

associated

with the NADH

N, as proposed by Ohnishi

centers

associated

as Centers S-l and S-2 (11). centers;

776

(4).

dehydro-

EPR signals

namely, Center N-2 (Fig.

in both reconstitutively

NADH dehydrogenases.

and Pring

with succinate

2), Center N-3 plus N-4 (Fig.

4); can be resolved (Spectra

iron-sulfur

l),

3) and Center N-5 active

(Spectra

A)

BIOCHEMICAL

Vol. 56, No. 3, 1974

AND BIOPHYSICAL

-1891?V 9°K

Figure

1:

t 193

EPR spectra of iron-sulfur center N-2 in the purified, reconstitutively active and inactive NADH dehydrogenases, respectively. Reconstitutively active (Fig. 1A) or inactive (Fig. 1B) NADH dehydrogenase (each at protein concentrations of 10.6 mg per ml) was stirred in 0.6 M sucrose, 1 mM histidine, and 50 mM Tris-HCl (pH 8.0) under a continuous flow of argon. The redox potential of the suspension was measured according to Dutton (9). The following redox mediators were added; 62.5 PM phenazine ethosulfate, 62.5 ~.IMphenazine methosulfate, 6.3 uM duroquinone, 6.3 uM pyocyanine, 7.5 nM resorufin, 30 PM 2 OHnaphtoquinone, 77.5 uM phenosafranine, 73.5 ~.IMbenzyl viologen, and 133 uM methyl viologen. The desired redox potential of the suspension, as shown in the Figure (-189 mV) was attained by the addition of aliquots of freshly prepared dilute solution of dithionite. Aliquots (about 0.3 ml) were transferred anaerobically to quartz EPR tubes and frozen rapidly by immersion in isopentane at its freezing point (113'K). EPR measurements were performed with a Varian E-4 spectrometer. Samples were cooled to 9°K with a fast stream of cold helium gas derived from boiling liquid helium. Temperature was measured with a thermocouple Au/Co versus Pt. EPR operating conditions; microwave frequency, 9.09 GHz; modulation amplitude, 12.5 gauss; microwave power, 10 mW; time constant, 0.3 set; Spectra A and B were recorded consecutively, scanning time, 500 gauss/min. using samples in EPR tubes of the same diameter.

In order to obtain minimum interference tials

RESEARCH COMMUNICATIONS

EPR spectra

from other

of individual

iron-sulfur

(Eh) of the enzyme suspension

iron-sulfur

centers,

appropriate

and the temperature

centers with redox poten-

of EPR measurements

only one of two iron-sulfur N-l Footnote 1: Using pigeon heart mitochondria, value. This components was found to show an ATP-dependent midpoint potential component was designated as Center N-la, the other as Center N-lb, before individual EPR signals were resolved potentiometrically (13). More recently, the Em?,2 values of Center N-la and N-lb in pigeon heart mitochondria were determined as -380 +15 mV and -250 It 15 mV, respectively (4).

Vol. 56, No. 3, 1974

AJ

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2 03 X08

,94 -455mv 23 * K

- 34c 75-K

:;it 93

B

B X08

J

0&

2

V

31

33

35

Figure 2:

EPR spectra of iron-sulfur center N-la plus N-lb in the reconstitutively active (A) and inactive (B) NADH dehydrogenases, respectively. Experimental conditions were the same as described in Figure 1, except for the Eh value of the enzyme suspension (-455 mV) and the sample temperature of EPR measurements (23'K).

Figure 3:

EPR spectra of reconstitutively active (A) and inactive (B) NADH dehydrogenases, showing signals arising from Center N-3 plus N-4 Experimental conditions are the same as described in the legend of Figure 1, except for the Eh value (-340 mV) and sample temperature of the EPR recording (7.5'K).

were chosen, as illustrated

in the Figures.

individual

iron-sulfur

(Fig.

Spectra A) shows no significant

sulfur

l-4,

centers

iron-sulfur

centers

of the corresponding drogenase,

general

from that SMP (12).

inactive

centers

Most of the

in the reconstitutively

EPR characteristics

suggest that

around the iron-sulfide

of the iron-

NADH dehydrogenase

show

active

some modification centers

778

are quite

of molecular

is associated

activity.

active

dehy-

such as the g value of the signa IS

and power dependence of the EPR spectra

These observations

tutive

or in beef heart

dehydrogenase

1 - Fig. 4, Spectra B), in comparison with those

iron-sulfur

although

and temperature

(Fig.

active

differences

in the reconstitutively

broadened EPR spectra

shape of EPR spectra of

in the reconstitutively

in Complex I (7,12)

centers

The line

similar.

envimnoment

with the loss of reconsti-

Vol. 56,No.:3, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

A --+lf 2t II

XI

t I I.89 1.90

EPR spectra of reconstitutively active (A) and inactive (B) NADH dehydrogenases, showing signals arising from Center N-5 plus N-6. Experimental conditions are the same as described in the legend of Figure 1, except for the Eh value (-455 mV), sample temperature of the EPR recording (SOK), and the EPR power setting (5 mW).

In Complex I, the measured Em8. 0 value of Center -135 f 15 mV, which is considerably for Center N-2 in beef heart

traction

of the dehydrogenase

becomes further the succinate stitutively

negative

active

as in the case of iron-sulfur

(11).

dehydrogenase

in the inactive

N-2

than the Em8 0 value

SMP (Em8 . O = -80 mV f 15 mV). Upon exfrom Complex I, the Em8 o value of Center N-2

electronegative, dehydrogenase

is approximately

N-2

more electronegative

obtained

Center

COMMUNICATIONS

1 207

-455mv 5'K

Figure 4:

RESEARCH

Center S-2 in

The Em8. o value of Center N-2 in the reconis -210 f 15

dehydrogenase

mV,

while

the Em8. o value of

shows a tendency to go further

electro

(-265 + 15 mV).

Iron-sulfur was resolved

Center N-l, potentiometrically

contributing into

two components'

using a computer program devised by Martin N-la and N-lb

to the so-called

(14).

[Center N-la and N-lb],

Em8. o values of Center are -385 t 15 mV and -260 f 15 mV, respectively. Midpoint

779

Pring

"g = 1.94" signal,

Vol.

56,

No.

potentials tion

of

of the

Center

BIOCHEMICAL

3, 1974

these

Centers

enzyme

from

seem to remain

Complex

EPR signals

which

I,

resulting

ved potentiometrically. value

of

ase,

which

-245

almost

RESEARCH

unchanged

differs

ected

from

similar

ref.

helium

(Figure

arises

from

and 1.89 N-3 nor

the

COMMUNICATIONS

during

the

extrac-

characteristics

N-4.

Under

of

centers,

for

midpoint

active

and inactive

purified -135

extraction dramatic

of the midpoint

These

data

a phospholipid to a more to shift

the

Em8 . o value

potential

change that

in the

about

iron-sulfur

out

(4))

from

the

differ-

spectra

of

reconstitutively I.

60% of

Center

do not

with

N-2 is

in Complex

centers

but

raised

I before

show such

the a

upon reconstitution. center

N-2

membrane,

by removal

active

Center

NADH dehydrogenase

originally

mitochondrial

potential of the

of

g = 2.11

two different

combined

in Complex active

iron-sulfur

environment

of the

of liquid which

and N-6

N-52

been det-

neither

at least

-260 mV in both

obtained

Other

midpoint

Upon recombination

of

and also

enzyme).

layer aqueous

region

have

Thus,

be sorted

Em8 . o values

of reconstitutively

suggest

from

More recently,

signal

from

an Em8 . 0

dehydrogen-

to that

g = 1.87

arise

cannot

the Em8 o value

+ 15 mV (the

and 1.89 close

as Centers

give

I.

undetectable.

to arise

two centers

in the

phospholipids,

the

almost

appear

dehydrogenases,

Upon recombination

1.90

low temperatures

potentials.

N-S and N-6 were

is

are designated

these

Complex

low temperatures

and N-4,

signals

which

2.07,

been resol-

or inactive

from

EPR conditions,

at such

four

responsible

Centers

N-3

obtained These

in their

these

obtained

not

signals

active

of 2.11,

at extremely

and N-4 have

combined

reconstitutively

at g values

Centers

signals

iron-sulfur signals

4).

N-3

of the

Em8 0 value

to the

(4,15)]

both

Center

Redox titrations

EPR signals [c. f.

from

f 15 mV in either

is

additional

to

BlOPHYSlCAi

N-2. So far,

ence

AND

of phospholipid

of Center

N-2 to

dehydrogenase

is probably and exposure with

surrounded of the cholate

phospholipids,

center tends

a more electronegative

with

by

value. the

shift

Footnote 2 : Center N-5 is not related to the previously reported “Center 5”. “Center 5” which shows EPR signals with g values of 2.09 and 1.89, was detected in cytkhrome bcl particles (16)) but not in NADH dehydrogenases.

780

Vol.

56,

No.

3, 1974

in midpoint

BIOCHEMICAL

potential

is partially

stitution

procedure,

onment.

The fraction

the extent

part

centers

This enzyme contains

either

K3Fet:CN)6 or UQ as electron

original

be reported

agreement with Detailed

dehydrogenases

elsewhere

(12).

a reconstitutively

active

from mitochondrial

almost no phospholipids The latter

acceptors.

envir-

NADH

membranes with

and reacts reaction

with

is inhibited

about 30% of Center N-2 in the NADH dehydrogenase

that

X-100 has an Em8 o value almost the same as in Complex

with Triton

I (-135 5 l!< mV), although

it

shows quite

broad EPR spectra

of Center N-2 in the reconstitutively communication

ModifiN:ations environments, of EPR spectra quite

to its

or Amytal.

It is interesting

in this

during the recon-

NADH-UQreductase.

which was extracted

X-100.

that

that

in Complex I, in purified

Baugh and King (17) reported preparation

COMMUNICATIONS

(60%) is in reasonable

systems, will

Triton

solubilized

RESEARCH

suggesting

of rotenone-sensitive

and in the reconstituted

by rotenone

reversed,

which is restored

data on these iron-sulfur

dehydrogenase

BIOPHYSICAL

of the Center N-2 is restored

of restoration

Recently,

AND

(Figure

of active

independent

similar

to

NADH dehydrogenase described

1, spectrum B).

of the active

give rise

inactive

(18,19)

center

to either centers;

of iron-sulfur

changes in midpoint

proteins

or of their

potentials,

however, these two parameters

and the molecular

or broadening appear to be

mechanism for these changes remains to be

elucidated.

ACKNOWLEDGEMENTS The authors his interest

would like

to express their

and encouragement

Miss S. Shiraishi

throughout

for her excellent

This investigation

was partly

this

technical supported

CA-08964.

781

gratitude work.

to

Dr.

B. Chance for

Thanks are also due to

assistance. by PHS grants

GM-12202 and

Vol.

56,

No.

3, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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REFERENCES

Ragan, C. Ian and Racker, E. (1973) J. Biol. Chem. 248, 6876-6884. Hatefi, Y., Haavik, A.G. and Griffiths, D.E. (1962) J. Biol. Chem. 237, 1676-1680. 3. Hatefi, Y., Haavik, A.G. and Griffiths, D.E. (1962) J. Biol. Chem. 237, 1681-1685. 4. Ohnishi, T. and Pring, M. (in press) Dynamics of Energy Transducing Membranes, Elsevier Publishing Co., Amsterdam. 5. Ohnishi, T., Asakura, T., Yonetani, T. and Chance, B. (1971) J. Biol. Chem. 246, 5960-5964. 6. OhGhi, T., Wilson, D.F., Asakura, T. and Chance, B. (1972) Biochem. Biophys. Res. Commun. 46, 1631-1638. 7. Orme-Johnson, H.R., Orme-Johnson, W.H., Hansen, R.E., Beinert, H. and Hatefi, Y. (1971) Biochem. Biophys. Res. Commun. 44, 446-452. 8. Albracht, S.P.J. and Slater, E.C. (1971) Biochim. Biophys. Acta 245, 503-507. 9. Dutton, P.L. (1971) Biochim. Biophys. Acta 226, 63-80. 10. Wilson, D.F., Erecinska, M., Dutton, P.L. and Tzudsuki, T. (1970) Biochem. Biophys. Res. Commun. 41, 1273-1278. 11. Ohnishi, T., Winter, D.B., Lim, J. and King, T.E. (1973) Biochem. Biophys. Res. Commun. 53, 231-237. Ohnishi, T. and Ragan, C. Ian, in preparation. 12. 13. Ohnishi, T., Wilson, D.F. and Chance, B. (1972) Biochem. Biophys. Res. Commun.,49, 1087-1092. 14. Dutton, P.L., Erecinska, M., Sato, N., Mukai, Y., Pring, M. and Wilson, D.F. (1972) Biochim. Biophys. Acta 267, 15-24. 15. Albracht, S.P.J. (in press) for Dynamics of Energy Transducing Membranes, Elsevier Publishing Co., Amsterdam. 160 Ohnishi, T., Winter, D.B. and King, T.E. (1973) Fed. Proc. 32, 595. 17. Baugh, R.F. and King, T.E. (1972) Biochem. Biophys. Res. Commun. 2, 1165-1173. 18. DerVaranian, D., Baugh, R.F. and King, T.E. (1973) Biochem. Biophys. Res. Commun. S& 629-634. 19. Ohnishi, T., Baugh, R.F. and King, T.E., unpublished observation. 1. 2.

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