A 17O-effect on the EPR spectrum of the intermediate in the dioxygen-laccase reaction

BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A I7 O-EFFECT ON THE EPR SPECTRUMOF THE INTERMEDIATE IN THE DIOXYGEN-LACCASE REACTION

R. Aasa,

Department of Biochemistry of Ggteborg and Chalmers Institute of Technology Fack, S-402 20 GiSteborg 5, Sweden

University

Received

R. Brand&, J. Deinum, B.G. Malmstrijm B. Reinhammar and T. V;inng&d

April

14,1976

SUMMARY: I7 O-enriched 0 has been used to show by EPR spectroscopy that the paramagne ?-ic intermediate, which is formed when anaerobically reduced fungal lactase reacts with dioxygen, is a true oxygen intermediate. Microwave power saturation studies at 4.2 K further emphasize its unusual relaxation properties. Reoxidations were also performed at different pH values and revealed that the paramagnetic intermediate can exist in two different forms in the lacquer tree lactase.

The mechanism of those and lactase,

which

much attention certed other

(1,2).

both

Rather

four-electron

after

transfer

demonstrated

a long

with

however,

difficult

species

observed,

but rather

to assign

reflect

changes

(3,4).

Only

intermediates

c oxidase

and lactase

electronic

structures

in the

Any

intermediates.

have optical

may not

con-

even involve properties

of

been

(5-7). to

oxygen

It

is,

optical directly,

enzyme-bound

me-

ions. Recently

mediate

we demonstrated

in the reaction that

this

teracting

with

a paramagnetic

0 1976

by Academic Press, in any form

of reproduction

represents

Inc. reserved.

an EPR signal

of reduced

suggested

Copyright All rights

mechanisms involving

enzyme-bound

cytochrome

c oxidase

atoms of 02 to H20 have attracted

have been suggested

search,

and they

such as cytochrome

unlikely

mechanism must involve

recently,

tal

reduce

enzymes,

lactase

an oxygen metal 1204

of a paramagnetic with radical,

0

and it 2' possibly

ion in the enzyme (8).

interwas inIn

Vol. 70, No. 4, 1976

BIOCHEMICAL

0.35

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 0.39

0.37

Fig. 1 (A). High-field part of the EPR spectrum of fungal lactase A, recorded at 9 .I4 GHz. The enzyme was anaerobically reduced with four electron equivalents of ascorbic acid and mixed with 0 -saturated buffer, 100% 160 and 91.8% 170, respectively. The geaction was quenched after 30 ms. The protein concentration was 270 ~.IMin 50 mM Na acetate buffer at pH 5.5. The spectra were recorded at 16 K and a microwave power of 170 mW, which are non-saturating conditions for the intermediate signal. (B). Simulated spectra of the signals in (A) assuming rhombic g-tensor (gX=1.906, gy=1.939, g,=2.16) and anisotropic l'0 hyperfine splitting. The "0 spectrum (91.8% "0, I=5/2 and 8.2% 160, I=O> was simulated assuming interaction with one "0 nucleus with A =5.5 mT and A =A =O. Lorentzian line-shape and a linewidth of"15 mT were us3d.s this

communication

substituting this

signal

we report

1702 for involves

1602,

an effect thus

providing

at leastoneatom

oxygen

molecule.

We also

studies

further

illuminating

present

on this

EPR signal evidence

direct

of 0 derived

some microwave

the unusual

by

from the

that di-

power saturation

properties

of the

inter-

mediate. MATERIALS AND METHODS: Fungal and lacquer tree lactase were prepared as described previously (9,101. The anaerobic and rapidfreeze techniques were the same as used earlier (6). The EPR specfya were recorded at 9.14 GHz in a Varian E-9 spectrometer. The O-enriched 0 (91,8%) was purchased from Miles Laboratories Ltd, Slough S22 4LY, England. A device was construcf?d permitting the addition of controlled small amounts of the O-enriched O2 to buffer solutions.

1205

BIOCHEMICAL

Vol. 70, No. 4, 1976

RESULTS AND DISCUSSION: aerobically signal

reduced

is

linewidth

at half

25 mT in the the

signal

tion

lactase

When I? O-enriched

which

with

EPR

02 is used the

increases

unambiguously

an oxygen

and an-

an intermediate

of the maximum amplitude

(8)

does not

by 28% to

demonstrates

intermediate

that

in the reduc-

lack

of effect

on the

signal

when

of the much smaller 17 detection of a 0-hyperfine

permit

17

O-enriched

enrichment

(40%)

splitting

less

6 mT.

strong

shape of the

0 hyperfine

difficult.

splitting

However, 17

substitution

of

by a shift

of the

earlier

it 0

sor giving

(81,

signal

both rather

splittings

in Fig.

IB (anisotropic

spectra

fit

the

spectra.

the

from

is not

absence

of such a shift

With

part

the

oxygen

a coupling

the

corresponding

and

given simulated experi-

we have assumed

largest

of about

and the

parameters

O-spectrum

g-ten-

of g-values

tensors),

of the

17

with

the

the

be correct.

combinations

g- and hyperfine

simulated

means that

can not

the

accompanied

has an isotropic

5.5 mT. In case of an interaction

atoms of

1A that

signal

special

one oxygen

Fig.

This

line,

non-overlapping

with to

the

with

rather

field.

seem necessary.

For the

an interaction equal

that

simulations

I6 0 (I=01

derivative

to reproduce

hyperfine

for

(1=5/2)

by overlap

makes a determination

by spectra

can be observed

a symmetric

shape of the

mental

is obscured which

peak to higher

assumption

In order

signal

Type 1 Cu2+ absorption,

17

of the

valent

lactase

dioxygen

to water.

The overall

ting

between

was used was a result

which

the

reaction

or tree IA).

is associated

The earlier

than

(Fig.

fungal

of oxygen

oxygen

In the

fungal

observed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hyperfine with

3.5 mT to

split-

two equieach atom

is required. It

can be noticed

that

both

the

1206

estimated

magnitude

and an-

BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4.2 9.14

K

Gtlz

1

0.5

0

I

I

I

1

I

1

2

3

4

5

LOG

MICROWAVE

POWER

( JJW)

curves of the intermediate Fig. 2. Microwave power saturation signal in the reduction of ox gen by reduced fungal lactase I60 (V) and 91.8% with 100% ' Y0 (o), respectively. The spectra were recorded at 4.2 K. The signal height used for the ordinate is the maximal derivative signal amplitude, measured in arbitrary units at about 0.345 T.

isotropy

17

of the

as reported

0 hyperfine

earlier

for

The saturation Fig. the

existence

free

possibility ion

1

Cuzt

in tree

(7)

expect

even if

enzyme.

unusual

Type

which

netic

relaxation

time

excited

radical

states.

of the

oxidized (21,

This

with

on the

state.

1207

a paramagnetic of the

are observed

However, with

of the

for

Another

(15).

signal (8).

by

unusual

EPR properties

proteins

could

is

property

lactase

behavior

shown in

be explained

an interaction

relaxation

intermediate

could

intermediate

if

same range

(13,141.

interacts

of the

in the

at 4.2 K is

O- can have this

changes

Cu2+-Cu2+ pair

in the

signal

as in fungal

in the

are

and O-

of the

Small changes

as well

caused the

magnetic

the

in presence

more drastic

3 Cu

(11,121

low-lying

is that

in the

Type

short

of

radicals,

0;

behavior

2. The very

splitting

one would

this

oxygen

copper

radical.

exist

as a non-para-

other

hand be paramag-

BIOCHEMICAL

Vol. 70, No. 4, 1976

Reoxidation also

of reduced

studied

mediate a signal width

with

were observed

signal

dence of the

pH-dependence

that

ted and unprotonated the

oxygen

spectrum. effect

the

More extensive

radical

was

interAt pH 4.5

At pH 7.4 the

line-

on the

fungal

explanation

groups

shape of the

with

can exist

of course,

studies

line

enzyme.

be detected

A possible

Titrating

can,

of the

the tree

could

oxygen

forms.

lactase

to 75 mT. No such pH depen-

signal

intermediate

of D20 on the

Two forms

for

has increased

pH 4.0 and 7.5.

is

tree

of 40 mT appears.

intermediate

between

and lacquer

pH values.

a linewidth

of the

lactase

fungal

at different

EPR signal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

also

for

in protona-

in the

vicinity

of

its

EPR

influence

pH dependence signal

this

and the

at lower

pH have

been initiated. The present

work gives

of

an oxygen

radical

its

unusual

properties

a definite

identification

conclusive

intermediate it

evidence

for

in an oxidase.

has been suggested of the radical

will,

the

existence

On the

basis

to be O- (81, however,

of

but

require

much more work. ACKNOWLEDGEMENTS:We are indebted to Miss Ann-Cathrine Carlsson, who cultured the fungus and prepared the lactase. This study has been supported by grants from Knut and Alice Wallenberg Foundation and the Swedish Natural Science Research Council. REFERENCES: 1.

2. 3. 4. 5. 6.

Malmstrsm, B.G.,(1973) Q. Rev. Biophys. 6, 389-431. MalmstrGm, B.G., Andrgasson, L.-E. and Reinhammar, B. (1975) in: The Enzymes, (Bayer, P.D., ed.), Vol. 12B, pp.507-579, Academic Press, Inc., New York. Curzon, G. and Cumings, J.N. (1966) in: The Biochemistry of Copper, (Peisach, J., Aisen, P. and Blumberg, W.E.,. ehs.) PP.555, 578-582, Academic Press, New York and London. Ehrenberg, A. and Vanneste, W. (1968) in: Biochemie des Sauerstoffs, (Hess, B. and Staudinger, Hj. eds.) p. 128, Springer-Verlag, Berlin, Heidelberg, New York. Chance, B., Saronio, C. and Leigh, Jr., J.S.(1975) Proc. Nat. Acad. Sci.(USA)72, 1635-1640. Andrgasson, L.-E., Brbndgn, R., Malmstrijm, B.G. and V;inngdrd, T. (1973) FEBS Lett. 32, 187-189.

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Vol. 70, No. 4,1976

7.

8. 9. 10. 11. 12. 13.

14.

AND BIOPHYSICAL

And&asson, L.-E., Brand&, R. and Reinhammar, B. (1976) Biochim. Biophys. Acta, in press. Aasa, R., Br;ind&, R., Deinum,J., MalmstrCim, B.G., Reinhammar, B. and Vdnngbrd, T. (1976) FEBS Lett. 61,115-119. Brand&n, R., Malmstram, B.G. and V;inngbd, T. (1971) Eur. J. Biochem. 18, 238-241. Reinhammar, B. (1970) Biochim. Biophys. Acta 205, 35-47. Tenth, A.J. and Holroyd, P. (1968) Chem. Comm., 471-473. Bray, R.C., Pick, F.M. and Samuel, D. (1970) Eur. J. Biothem. 15, 352-355. Brailsford, J.R., Morton, J.R. and Vannotti, L.E. (1968) J. Chem. Phys. 49, 2237-2240. Wong, N. -B. and Lunsford, J.H. (1971) J. Chem. Phys. 55, 3007-3012.

15.

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W.

(1964)

Naturwiss.

1209

51,

404.