Enhanced superoxide dismutase activity of pulsed cytochorme oxidase

Vol.

136,

No.

April

14,

1986

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

ENHANCED SUPEROXIDE DISMUTASE ACTIVITY Ali Department

Naqui’

433-437

PULSED CYTOCHORME OXIDASE

OF

and Britton

Chance

of Biochemistry and Biophysics, D 501 University of Pennsylvania, Philadelphia,

Richards Building, PA 19104

and Institute

Received

for Structural and Functional Center, 3401 Market Street. March

Studies, University City Philadelphia, PA 19104

Science

3, 1986

Summarv. The superoxide

disumutase (SOD) activity of beef heart cytochrome oxidase, both in the resting (as isolated) and pulsed (reduced and reoxidized) states, has been investigated using their ability to inhibit the autoxidation Resting oxidase showed variable SOD rate of pyrogallol and epinephrine. activity, while in the pulsed state the SOD activity of cytochrome oxidase (CcO) increased by an order of magnitude. These results are discussed in 0 1986 Academic Press, Inc. terms of a physiological role for the pulsed oxidase.

Introduction.

Cytochrome

E.C.

is

It

1.9.3.1)

the

four

in

metal

ions

and two associated

oxidase

electron

which

(ferrocytochrome

turn in

copper

oxygen

transfer

drives

02 oxide-reductase

to water

produces

respiratory in a four

a proton

) Recently

it

across

Cc_0 contains

two hemes (cytochromes

(Cu, and Cu,

chain.

electron

gradient

of ATP [Il.

the production

the molecule:

atoms

c:

of the mitochondrial

of molecular

This

process.

membrane,

at least

terminal

the reduction

catalyzes

transfer

the

2 oxidase

5 and a3)

has been

-3 reported

that

to be in the

Cc0 contains “resting”

and reoxidized Okunuki Antonini

et al

[4,5]

’ Correspondence Abbreviations: Resonance.

oxygen

and it [6,i’]

states

and Mg [31 as well.

The resting

state.

by molecular

dr Brunori

and reoxidized

Zn [2]

was termed

from

a kinetic

author

the difference

at the University

CcO: cytochrome E oxidase. SOD: Superoxide Dismutase

between the

of Pennsylvania

EPR: Electron

Ail

study

and coined

is

said

by dithionite

“oxygenated”

of intensive

perspective

433

once reduced

as the

has been a matter have approached

to this

oxidase

Cc0 as isolated

oxidase since

by

then.

the resting term

npulsed.W

address.

Paramagnetic

Copyright 0 1986 rights qj’ reproduction

0006-291 X/86 $1.50 by Academic Press, Inc. in any ,fbrm reserved.

Vol.

136,

No.

1, 7 986

BIOCHEMICAL

They found that oxidation

the pulsed oxidase

in the pre-steady

"pulsed"

state

state

is present

More recently adduct

AND

[9,10].

RESEARCH

has an enhanced rate region.

particles

oxygenated

of cytochrome

in the strict

peak moves to 420 nm (from 418 nm) instead

the traditional

"oxygenated"

form,

peak shifts

is indeed a peroxide (420 nm form) probably

variant

is more open than the resting

the physiological

role

(if

dismutase

(SOD) activity,

and thermal in the resting

in the literature

and reoxidized

open conformation

of continuing

has a much higher

SOD activity

are discussed

state

428

nm for

the 428 nm form

state

oxidase

and most resembling

by several

that

groups,

is not known.

CsO has a small

We have studied and report

absence of

the pulsed

by cyanide,

states

The results

that

efforts

that

is inhibited

of CQO [13].

that

a structure

any) of such a pulsed

which

denaturation

with

of

a peroxide

of H202 to the 420 nm

of the resting

state

In spite

(g-12).

There are indications

pulsed

It has been proposed

is a conformational

of the peroxidases

state.

Upon addition

to 428 nm, thus establishing

adduct.

the

is in fact

H202, the Soret

the Soret

that

c

[8].

oxidase

reduced Cc0 is reoxidized

oxidase.

COMMUNICATIONS

They also reported

in Keilin-Hartree

we have shown that If fully

BIOPHYSICAL

azide.

superoxide

very high pH

the SOD activity

here that

of Cc0

the pulsed or the

compared to the resting

in terms of a physiological

relevance

of the

state.

&&&&s

and Methods. C&O from beef heart was prepared according to Yonetani or Volpe-Caughey [15] slightly modified in our laboratory. Specifically, we add 1 mM EDTA in all our buffers to exclude adventitious metal ions from All other reagents were of highest purity available the final preparation. commercially. The 420 nm pulsed form was prepared essentially according to [9,101. Stock solutions of pyrogallol (200 mM) and epinephrine (saturated solution) were prepared in 10 mM HCl. Epinephrine solution was prepared fresh every day and kept on ice in an air tight vessel during the experiment. The SOD activity was measured by two different methods: 1) autoxidation of pyrogallol [16,17] and 2) autoxidation of epinephrlne [18,19]. All spectra were run on a Johnson Foundation dual wavelength spectrophotometer. [14]

&g,&Jts

and Dism

autoxidation as a function oxidases

process

.

Cc0 in the resting

markedly.

In Fig.

of Cc0 concentration.

are somewhat different

1,

state the rate

The curves for

different 434

for

inhibits

the pyrogallol

of autoxidation the resting

preparations

is plotted

and pulsed

(not shown).

Vol.

136,

No.

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

\\;_

*

,/* Restmg

-7-2

34

[ Cytochrome

5

c Oudose]

6

X IO6 (M )

02

AN 50

AN 99

Fig. 1. Inhibition of initial rate of pyrogallol (final concentration 0.2 r&l) autoxidation by C&O in resting and pulsed forms. The product of pyrogallol autoxidation was monitored at 459 nm (reference wavelength 575 nm). 0.1~1 phosphate buffer containing 1mU EDTA. pH 8.2. Temperature 21°C. Fig. 2, Inhibition of epinephrine (final concentration 15 u!4) autoxidation Cc0 in resting and pulsed forms. The product of epinephrine autoxidation monitored at 480 mm (reference wavelength 575 nm). O.lH carbonate buffer containing Id4 EDTA. pH 10.0. Temperature 21’C.

However, clear

the difference

when the

as well.

2 that

cl31 added

than

used

the

that

M of cao.

results

is

increase

and full

also

50% decline

oxidase,

the

a decrease

inhibition

in

at which

differences

they

used

by reported 435

from are much

States

The authors

the SOD activity

of Cc_0 and

autoxidation

can be achieved to our

with

results.

the resting

(Fig.

Markosian

the epinephrine heterogeneity

at 2.5

oxidase. but

However,

rate

is et al tests,

x

We do not

a small

2).

of the autoxidation Although

clear

pulsed

we observed rate

is

SOD activity.

contradiction

further,

always

method.

in activity

can be obtained.

the condittons our

of

It

in epinephrine

rate

is

the autoxidation

2.

and the

determining

noted

was increased

inhibition

to reconcile

for

in direct

instead

Fig.

the resting

the 502 of the uninhibited

the Cc0 concentration

of pulsed

shown in

Cc0 has an intrinsic

were

that

This

are

states

C&O inhibits

case of pyrogallol

method

They reported

ducible

that

tetrazolium

“similar

even achieve

report

in the

and pulsed

compared.

between

has been reported

tests.” 10-T

here

is

The results

the difference

more pronounced It

the resting

same preparation

of epinephrine Fig.

between

by uas

in

quite [13]

When reprothe

case

dramatic did

not

we may be able

in the Cc0 preparations

Vol.

136.

No.

BIOCHEMICAL

1. 1986

The enhanced

[20,211.

pyrogallol

method

the resting used

states

plays

Now the question The natural reader.”

is

by “mistake”

or during

takes

order value

resting

state it

“mistake”

state.

oxidase

oxidizes

&j&g&%.

d’

and pulsed

~“1

Does pulsed

raised

turnover

an intermediate.

necessary

Zn

for

scavanger for

CqO? or “proof

peroxidatic

ion

is

to “take

care”

is

0.35

above.

pulsed

one of the

generated of this

is

of this

form

species,

can indeed

Indeed,

the radical

pulsed

produce

than

that

is

436

that is

one

for

than

the

relevance?

g = 2 (from

(by

the

pulsed

state

[6,10].

The answer

an attempt

pulsed

the

“slowern

because

the resting

are

form,

rate the

the

more

a more active

more superoxide

EPR signal

form

at a greater

paper

can be postulated

If

a

by the fact

much higher

faster

the

known having

still

The reason

have any physiological

The results

2.5% is

seems reasonable

times

from

by 50%.

enzymes

the

be generated

the

in SOD activity

ion.

the pulsed

enhancement

It

[23],

(from

as judged rate

“fastest”

whereas

intermediate)

p five

oxidase

be explained

a superoxide

will

about

the autoxidation

could

if

ion

oxidase

unknown.

it

The differrence

forms

to “trap”

cytochrome

the question

be able

the Cu atoms reported

superoxide

shows that

conformation

An eight-fold

is

is

the

of “2 x 10g Ff’s-’

superoxide

the question

if

between

question.

and 1121 (proposed

being,

SOD is

constant

or as a turnover

resting

[13]

to inhibit

that

easier

that

the recently

of a superoxide

and 2.5% for

can be the following:

probability

that

that

an intriguing

t&O should

resting

has a “closed”

making

difference

faster

for

of 5 x lo7

the resting

was proposed

is

in the

but see 241.

rate

between

open,

turnover,

consideration

formidable

is with

of C-q0 required

into

a second

possibility

of the SOD activity

method)

concentration

it

clear

may be due to the conditions

Whether

property

The reason

123,

QuantitatiOn Pyrogallol

[22]

also

in SOD activity

as to why a SOD activity

in accordance

activity).

substance

this

arises

and catalatic

toxic

in

and simple This

report

COMMUNICATIONS

Cc0 is

change test

in the SOD activity.

any role

RESEARCH

of the reoxidized

in epinephrine

In an earlier

involved

BiOPHYSlCAL

The more pronounced

1).

and pulsed

of Cc0 are atom [2]

SOD activity

(Fig.

(pH 10.0).

AND

to

to answer

oxidase-

being

by “mistake” superoxide)

a or as

seen

Vol. 136,

No.

during

1, 1986

turnover

discusion

of Cc0 is higher

see [261).

concentration reading”

BIOCHEMICAL

Whatever

AND

under *high

of pulsed oxidase

power”

turnover

RESEARCH

COMMUNICATIONS

conditions*

the reason may be. the higher

demands more *protective

or *higher

BIOPHYSICAL

devices*

to dismutate

may have a physiological

it.

and

more

C251 (for

a

superoxide

careful

*proof

Thus, the enhanced SOD activity relevance.

m. We thank Mr. E. Gabbidon and MS, K. Kozinski technical assistance and Dr. K-G. Paul for fruitful discussions. was supported by NIH grants IU31909 and GM33165.

for excellent This rork

References ~1. Wikstrom, M., Krab, K. and Sarastg K (1981) Cytochrome Oxidase: A Synthesis, Academic Press. New York. 2. Einarsdottir, 0. and Caughey. W.S. (1984) Biochem. Biophys. Res. Commun. 124, 3.

836-842.

Einarsdottir, 129,

0. and Caughey, W.S. (1985) Biochem. Biophys.

Res. Commun.

8408-847.

Orii, Y. and Okunukt, K. (1963) J. Biochem. (Tokyo) 53. 489-499. Proc. Int. Okunuki. K., Hagihara, B., Sekuzu, I. and Horio. T. (1958) Symp. Enzyme Chem. I Maruzen Tokyo, pp 264-272. E., Brunori. M., Colosimo. A., Greenwood, C. and Wilson, M.T. 6. Antonini. (1977) Proc. Natl. Acad. Sol. USA. 74, 3128-3132. M., Colosimo. A., Rainoni, G., Wilson, M.T. and Antonini. E. 7. Brunori, (1979) J. Biol. Chem. 254, 10769-10775. 8. Bonaventura, C., Bonaventura, J., Brunori, M. and Wilson, M.T. (1978) FEBS Lett. 85, 30-34. A. and Chance. B. (1984) J. Biol. Chem. 259, 2073-2076. 9. Kumar, C., Naqui.. 10. Kumar, C. , Naqui. A. and Chance. B. (1984) J. Biol. Chem. 259, 11668-11671. R.A., Naaqui. A., Chance. B. and Spiro. T.G. (1985) FEBS Lett. 11. Copeland, 4. 5.

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L. and Ching, Y-C. (1983)

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N.A. and Nalbandyan. R.M. Markossian, K. A. , Poghossian. A. A. , Paitian, (1978) Biochem. Biophys. Res. Commun. 81, 1336-1343. T. (1961) J. Biol. Chem. 236, 1680-1688. 14. Yonetani, Yoshikaua. S., Choc, M. G., O’Toole, H.C. and Caughey. W.S. (1977) J. 15. Biol. Chem. 252, 5498-5505. Marklund. S. and Harklund, G. (1974) Eur. J. Biochem. 47, 469-474. 16. Chance. B. and Maehly, A.C. (1955) in Methods in Enzymology (S.P. 17. Colowick and N-0. Kaplan, eds.) Academic Press, New York pp. 764775. 18. Misra, H.P. and Fridovich. I, (1972) J. Biol. Chem. 247. 3170-3175). 19. Sun. M. and Zigman. S. (1978) Anal. Bioohem. 90, 81-89. Naqui, A., Kumar, C., Ching, Y. C. , Powers. L. and Chance. B. ( 1984) 20. Biochemistry, 23. 6222-6227. 21. Brudvig, G.W., Stevens, T.H., Morse. R.H. and Chan. S-1. (1981) Biochemistry, 20, 3912-3921. 22. Markosyan. K.A. and Nalbandyan. R.M. (1978) Biokhimiya (Moscow), 43. 1143-1149 (Eng. translation pp 899-904, 1979). Fridovich. I. (1974) Adv. Enzymol. 41. 35-97. 23. Fee. J.A. (1980) in Metal Activation of Dioxygen (T. G. Spiro ad.) John 24. Wiley, New York pp 209-237. 25. Wilson, M.T., Jensen, P., Aasa. R., Malmstrom, B.G. and Vanngard, T. (1982) Biochem. J. 203, 483-492. 26. Naqui, A., Chance, B. and Cadenas, E. (1986) Ann. Rev. Biochem. 55:137-166 13.

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