Protocatechuate 3,4-dioxygenase. Resonance Raman studies of the oxygenated intermediate

Vol.

89,

No.

2, 1979

July

27, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

420-427

PROTOCATECHUATE 3.4-DIOXYGENASE. RESONANCE WN STUDIES OF THE OXYGENATED INTERMEDIATE

Oregon

William E. Keyes and Thomas M. Loehr* Department of Chemistry and Biochemical Sciences Graduate Center, 19600 N.W. Walker Road, Beaverton, Oregon Mary L. Taylor Department of Biology State University, Portland,

Portland

Oregon

97207

Oregon

97207

97005

and Joann Sanders Loehr Department of Chemistry State University, Portland,

Portland Received

June

4,197V

of protocatechuate 3,4-dioxygenase from SUMMARY: Resonance Raman spectra Pseudomonas aeruginosa have been investigated during the reaction of the enzyme with substrate and oxygen. It is found that the spectrum of the turned-over enzyme is indistinguishable from that of the resting enzyme in the absence of substrate, and is characterized b resonance-enhanced tyrosinate ring vibrational modes at 1263 and 1174 cm- P . In the ternary ES02 complex, however, the tyrosinate vibrational modes are shifted to 1252 and 1165 cm-l, respectively. There is no evidence for an dioxygen vibrations in the spectra of ES02 complexes prepared with 1602, Tao2, and 160180 in the region between 1300 and 200 cm-1. The results of this resonance Raman study are interpreted to indicate that molecular oxygen is attached only to the substrate (but not iron) in the stable intermediate, and that the concomitant rearrangement at C4 of the substrate induces a substantial change in geometry of the tyrosine residues associated with the iron complex. Furthermore, the optical spectrum of the ES02 complex (1 max = 520 nm) is dominated by tyrosinate + Fe(II1) charge transfer and contains little or no peroxide + Fe(II1) charge transfer. These results invalidate the previously advanced analogy in spectral properties between this enzyme and the respiratory protein, oxyhemerythrin. INTRODUCTION: tic

ring

structure pic

Protocatechuate

cleaving

Electron

[l].

studies

spin Author

//

Protocatechuate (decyclizing),

C’opyriRht All riRhrs

X/79/1

ternary

Fe(II1)

*

0006-291

has a molecular paramagnetic

on the resting

substrate-oxygen high

enzyme,

3,4-dioxygenase

complex

to whom correspondence

40420-08$01

3,4-dioxygenase EC 1.13.11.31

should

be

These

420

aroma-

and an 8(cc2@2Fe)

complex the metal studies

spectrosco-

(ES),

and enzyme-

ion occurs clearly

sent.

[protocatechuate:oxygen abbreviated 3,4-PCase

.00/O

@ 1979 by Academic Press, Inc. of reprodu4on in any form reserved.

[2,3].

, an intradiol

(EPR) and Mi;ssbauer

have shown that

species

is

of 700,000

enzyme-substrate

(ES02)

in each of these

weight

resonance

enzyme (E),

i/

(3,4-PCase)

3,4-oxidoreductase in this paper.

as

indica-

Vol.

89,

No.

2, 1979

ted changes nary

in the electronic

complex

peroxide

and led

lsince

erythrin.

iron

resonance

center.

[7,8]

unable

but

the metal--ligand

upon formation

the

iron

groups.

of the ter-

in ES02 may be coordinated similar

to that

of E [4-61

Subsequent

to

of oxyhemhave shown that

studies

on ES and endue to iron-

information

on the

the ES02 ternary

complex

by RR spectrosco-

rearrangement

occurs

in the iron-tyro-

structural

of the ES complex

any spectral

COMMUNICATIONS

new Raman frequencies

no further

upon reaction

to obtain

of the iron

have revealed

a significant

environment

RESEARCH

Raman (RR) investigations

or inhibitor,

that

BIOPHYSICAL

maximum at 520 nm is

We have now investigated

py and find

we were

that

to tyrosinate

complexes

substrate

sinate

to the proposal

is coordinated

zyme-inhibitor

AND

environment

the absorption

Recent

the metal

bound

BIOCHEMICAL

evidence

for

with

structure

molecular

the presence

of the

oxygen.

However,

of dioxygen

in

chromophore.

MATERIALS AND METHODS: Protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa was prepared as described previously [5] and had a specific activity >40 unitsfmg enzyme. The enzyme was dissolved at a concentration of %120 mg/mL in 0.05 M Tris-Cl (pH 8.5). For preparation of the ternary complex [9], the enzyme was stirred in the presence of 02 for 24 hrs. at 4°C. The enzyme solution was transferred to a capillary tube and the reaction initiated by addition of a small volume of a deoxygenated substrate, 3,4-dihydroxyphenylpropionate (pH 8.1). The contents were mixed with a syringe and frozen in liquid N2 within 15 s of the addition of substrate. The resulting trapped intermediate could be maintained at liquid NP temperature for several days with little or no decay as judged by its Raman spectrum. However, prolonged laser irradiation led to a slow degradation of the ES02 complex, permitting observation of the decay process. Ternary compiexes were alsor;ade with isotopically-enr:;hed dioxygen (Miles Laboratories): 02 (99 atom-% 0) and r601*0 (58 atom-% 0). Raman spectra were recorded on a computerized Jarrell-Ash spectrophotometer as reported elsewhere [lo]. The presence of a fluorescent background and the desire for spectra of high signalto-noise ratio necessitated the accumulation of approximately 300 scans per sample, each collected at a rate of 4.0 cm-'/s with 8 cm-l slits and a digitizing increment of 0.6 cm-l. Spectra of the resting enzyme (Amax = 465 nm) were obtained by excitation at 514.5 nm (~200 mW) while spectra of the ES complex (Amax = 480 nm) and by excitation at 530.9 nm (%50 the ternary complex (Amax = 520 nm) were obtained mW at the sample). RESULTS:

The Raman spectrum

to go to completion indistinguishable due to products (resting

is from

could

or turned-over)

of 3,4-PCase

after

the enzymatic

shown in the bottom

trace

that

enzyme

of the resting

be discerned. is

characterized

1263 and 1174 cm-1 due to tyrosinate

The 1300-700

of Figure [4-61

1.

reaction

is

This

spectrum

and no spectral

cm-1 spectrum

allowed

features

of the enzyme

by resonance-enhanced Raman peaks -1 ligand and at 755 cm due to tyrosine

421

is

at or

Vol.

89,

No.

2, 1979

BIOCHEMICAL

iI00

AND

BIOPHYSICAL

1060 FREQUENCY,

940

RESEARCH

COMMUNICATIONS

700

020 cm-’

Figure 1. Raman spectra of protocatechuate 3,4-dioxygenase in frozen solution after reaction with substrate (3,4-dihydroxyphenylpropionate) and oxygen. Upper spectrum: reaction with I8 02 (2 atm) to yield %75% ternary complex,ES02. Middle spectrum: reaction with I602 (1 atm) to yield %50% ternary complex. Lower spectrum: reaction with 1602 followed by complete decay of ternary ES02 complex into products at -185°C. Excitation at 530.9 nm (\50 mW at the sample). Data collection parameters are given in the text.

tryptophan protein dard, tyrosine

[6].

In addition,

the less

modes

enzyme with

nance-enhanced ternary

ES02

intense

peaks

sulfate

peaks

at 1002 cm-1 from

ion

present as an internal stan-1 at 864 and 833 cm are most likely protein

[ll].

In agreement resting

are non-enhanced

and 981 cm-1 from

phenylalanines while

there

with

substrate

vibrational complex

previous

results

alone

work

[4,7],

produces

modes of the iron in marked

changes

423,

we have found no significant protein.

that

reaction

changes However,

in the

formation

in the resonance-enhanced

of the resoof the spectral

Vol.

89,

lines

No.

2, 1979

attributed

tyrosinate

than

shifts

in

Fig.

1 from this

into to

to

tyrosinate

vibrational

frequency

In

BIOCHEMICAL

the

in

it

is

have

981

cm

-1

The

the

standard

intensity

unreacted

species

of

peroxide

or

three

by

and

have

cm

the

cm -'

which and

are which

heavier

oxygen

DISCUSSION:

Resonance

of

has

3,4-PCase

and

the

fer

(CT)

change

cm

have

the

top

-1

of

two

and

in

the

by

the

upper

cm

band

is

lower

in

the

in

are

upon

spectrum

of

of

both

form.

split

relative in

entirely

The

ES02

intensities

in

The

spectrum

spectrum

spectrum

-1

enzyme.

bands

bottom

trace).

middle

enzyme

lower

a difference

%lO

turned-over

tyrosinate

or

upper

or

the

. disappears

as

in

Fig.

1.

complete ES02

Fig.

for-

and

1 is

The

E.

due

to

~25%

at in

of

to

Raman

locate

spectroscopic

revealed

peroxide at

of

1107

3,4-PCase

not

also

shift

to

with

spectral

-l

and

[151,

failed in

to

the

and

spectra

the

frequency

contain-

740

features

of

metal-bound

850

cm

vibrations

Raman

any

isotopic

metalloproteins

metal-oxygen

spectra lower

due

between

reveal

the

appropriately

of

of

to

different

features

vibrations

examination

present

three

studies ion

cm

Close

prepared

betof

the

between

resting

1300

or

upon

turned-

substitution

isotope. Raman

of other

maximum

reaction

were

Previous

excitation

established

assignment

absorption

peaks

the

to -1

resting

half

1,

ES0 2 are

in

illustrated

3,4-PCase

[13,16].

complexes -1

enzyme

bic

755

cm

vibration

570

ternary 200

order

superoxide.

superoxide 500

of

in

dioxygen

[12-141,

over

at

755

complexes

dioxygen

bound

and

either

according at

the

that

in

cm

-1

COMMUNICATIONS

enzyme. Ternary

ween

in are

seen

than

RESEARCH

(Fig.

1165

bandwidths,

ES0 2 complex

remaining

ing

clearly

vibration

of

and

approximately

greater

tyrosine/tryptophan mation

1252 modes

which

BIOPHYSICAL

tyrosine/tryptophan

frequencies

a sample

doublets,

at

corresponding

vibrational

spectrum

the

and

modes

the

AND

the

the

presence

visible

E with

1471,

1338,

lthe

frequencies

1317,

is

1269 the

cm-I

tyrosinate

the

the

an

absorption iron

The 465

shift

nm to

appearance

of [7,8],

modes.

[4-71

charge in

480

band

ligand

+ Fe(II1)

RR spectra

vibrational

47-3

as

[5,17].

by in

nm visible

tyrosinate

CT from

accompanied

465

tyrosinate to

-+ Fe(II1)

and of

the

proteins

tyrosinate S [9]

of

absorption

iron-tyrosinate for

within

the

apparent

nm upon new

trans-

anaero-

substrate

but

without

These

results

a

-1

Vol.

89,

provide

No.

2, 1979

direct

change

BIOCHEMICAL

evidence

in the optical

substrate with

substrate

absorption

+ Fe(II1)

longer

for

Conversion

coordination

which

excitation

absorption

maximum at 520 nm in ES02 [9]

vibrations

changes lence

spectra

structural

of the iron

strate

to iron

are

tional

frequencies

unlikelihood

of tryptophan

center.

not

assignment

tains tion

A disruption of the

to the intense

likely

effects

that

the

these the va-

of binding tyrosinate

subvibra-

it

the 13-L-hydroperoxolinoleic

have located peak upon

tyrosinate

ring

[3]

that

near

the

of ES02 also

755 cm-l [19]

with

involvement

vibration

in

the iron-

of tryptophan

interaction

as

resulting

geometry

-,

complex

424

the ES02 complex

of the similarity

CT transitions

[Fe(III)(EDTA)02] acid

peak to

in ES02 would

peak in ES0 2'

on the basis

-+ Fe(II1)

a tryptophan

the

modes at 1252 and 1165

interaction

coordination

Although of this

formation

of the

the direct

iron-tyrosinate

[6].

the assignment

of the tryptophan-tyrosine

moiety

[ZO];

[18]

755 cm-'

requiring

has been proposed

022-

favors

stacking

in the Raman spectrum

or tryptophan

The enhancement

of the 755 cm-l

a ferric-peroxide

1150 M-lcrn-l/Fe

tyrosine

the

by a ring

without

the loss

Recently

+ although

in ES02 since

to perturb

ligand

since

in intensity.

chromophore

for

to either

as an iron

be explained

ligand.

The visible

vibrations,

is

mode at 755 cm-l

of the

from the distortion account

sufficient

The disappearance

do not decrease

an iron

center

and the electronic

measurements

-'

tyrosinate

It

the

to tyrosinate

ring

in frequency.

fluorescence

a tryptophan

E and ES could

of a

in both

moiety.

tyrosinate

vibrational

be assigned

favors cm

the

in ES.

could

iron

[Z]

changes

be assigned

at the iron

constant

by themselves

of 3,4-PCase

mode,

that

the contribution

iron-tyrosinate

can still

~10 cm-l

The resonance-enhanced

a tyrosine

of the

alterations

remains

from

by marked

of resonance-enhanced

have decreased

reflect

and suggest

the RR modes of S, particularly

accompanied

and the vibrational

these

of ES arises

COMMUNICATIONS

[8].

of ES to ES02 is

CT on the basis

RESEARCH

to Fe(III)

enhances

electronic

Fe(II1)

BIOPHYSICAL

spectrum

CT transition

wavelength

AND

of 3,4-PCase of its

of oxyhemerythrin, es20 nm = 530 M-lcm-l/Fe

of lipoxygenase-1,

con-

520 nm absorpssoO nm = [21];

and

r+7,, nm = 1000 M-lcrn-l/

Vol.

89,

Fe

N’o.

[ZZ].

If

would

account

2800

M-Icm-I/Fe

tional

mode.

served

in

that

2,

an

iron-peroxide

RR

[9])

and

1300

iron

of

considerable

-

is

the

no 700

sumably,

it

represents

dioxygen

to

form

termediate

cm-'

experiments

present

the

peroxide

as

view

in

that

ES02

structure

in

absorption

spectrum

is

the

is

substrate

ob-

unlikely

complex.

a

the

na-

characteristic

intermediate. has

I)

Hamilton

already

Prereacted

or

a more

[23].

An

with

advanced

in-

intermediate

I’o0

0

O-0

-

;e

(11

such

as

structure

ES02

complex

II [24]

(11)

is

since

it

B-carboxyethylmuconic ture

I.

This

intermediate

or carbon

II,

question

and the from

the

use

reaction planar

of in

be

be

detected, of

several oxygen ES

recent

product

might be

by

would

acid

could

quencies

favored

to

studies

more than

on

likely

to

would

an

decay

if

RR

possibly

by

investigating

at tetrahedral

C4

spectral

the in

425

substrate ES02.

of

spontaneously

to

due

For

Assuming

the to

to

a broader

would

the

analogous lines

wavelengths. of

denaturation

intermediate

resolved

excitation

the

=

vibra-

on

of

as

nm

was

information

reaction

by

(E,-~~

it ES02

appearance

(such

it

moiety

the

direct

distinct

proposed

band

ES02,

in

the

a

3,4-Pease,

peroxide

of

no

ES02,

of

dioxygen

superoxide

which

II,

nm

a bound

intermediate

anhydride,

complex

COMMUNICATIONS

resonance-enhanced

yielded

a peroxide

the

a

RR

or

intermediate

a

to

ES02

520

to

of

the

either

such

region to

the

the

rise

RESEARCH

the

attributable

coordinated

supports

in of

give

vibration

BIOPHYSICAL

present

portion should

enzyme-bound

spectrum

AND

were

a

Since

Although ture

BIOCHEMICAL

for

the

the

1979

the range

either

struc-

bound of

structure

have

converted

that

the

freI

this substrate

is

Vol.

firmly would explain

89,

No.

2, 1979

held very

BIOCHEMICAL

in position likely

AND

by the enzyme,

result

the decreased

in changes vibrational

BIOPHYSICAL

RESEARCH

the rearrangement

of substituents

at C4

geometry

thereby,

in the iron-tyrosinate frequencies

COMMUNICATIONS

in the RR spectrum

and,

of ES02 [25].

ACKNOWLEDGMENT: This work was supported by a research grant from the U.S. Public Health Service, National Institutes of Health (GM 18865) for which the authors are very grateful. We (T.M.L. and J.S.L.) also thank the California Institute of Technology for its hospitality and members of the Chemistry faculty for enlightening discussions of portions of this work.

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Tatsuno, Y., Saeki, Y., Iwaki, M., Yagi, T., Nozaki, M., Kitagawa, T., and Otsuka, S. (1978) J. Am. Chem. Sot. 100, 4614-4615. Keyes, W.E., Loehr, T.M., Taylor, M.L. (1978) Biochem. Biophys. Res. Commun. 83, 941-945.

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