Formation of porphyrin π-cation radical in myoglobin a study on one electron oxidation products of nickel(II)-substituted hemoproteins

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1319-1325

Vol. 151, No. 3, 1988 March 30, 1988

A

FORMATION OF PORPHYRIN ~-CATION RADICAL IN MYOGLOBIN STUDY ON ONE ELECTRON OXIDATION PRODUCTS OF NICKEL(II)S U B S T I T U T E D HEMOPROTEINS Isao Morishima

, Motoru

Division Graduate School

Takeda,and

Kikuo

Takatera

of Molecular Engineering, of Engineering, Kyoto University Kyoto 606, JAPAN

Received February 8, 1988

SUMMARY: N i c k e l ( I I ) - s u b s t i t u t e d m y o g l o b i n (Mb}, h e m o g l o b i n (Hb) and h o r s e r a d i s h peroxidase (HRP) were oxidized with iridate to examine whether porphyrin ~-cation radical is formed or not in these hemoproteins. It was found that N i ( I I ) - p o r p h y r i n ~-cation radical is formed in all of these hemoproteins as confirmed by UV-visible and ESR spectra, although the porphyrin ~-cation radical in Mb and Hb was less stable than in HRP. These results are discussed in relation to the different features of higher oxidation states of native Mb and HRP. ©1988AcademicPr.... Inc. The has

porphyrin

been

because

the subject it

(oxo-ferryl reaction 450

II)

and

cycle

electron

Oxidation

~

accompanied

with

oxidation

oxidized

(5).

compound

peroxidase

as

(CCP)

a free radical

It is still

* To whom correspondence

(Mb)

should

and

(Fe(IV))

last

well

as

residing

on

dec~de,

Compound

the

hemoglobin

ferryl the

P-

(Hb)

(compound

experiences compound

I

enzymatic

cytochrome

porphyrin

(HRP)

produces

puzzling

in

and possibly

peroxidase I

hemoproteins

intermediate

catalase

ferryl

in

for the

radical)

of myoglobin

while h o r s e r a d i s h

cytochrome

studies

~-cation

of peroxidase,

oxidized

(Po t. ) formed

as a reaction

porphyrin

one-electron

(4),

radical

of intensive

is involved

(I-3).

afford

z-cation

two-

II

and

porphyrin

protein

why a Po ÷" is not

upon

formed

in

be addressed.

Abbreviations: Mb, myoglobin; Hb, hemoglobin; HRP, horseradish peroxidase; CCP, cytochrome ~ peroxidase; Po, porphyrin; Po +', porphyrin T-cation radical; PP, protoporphyrin; DiAcP, diacetyldeuteroporphyrin; Py, pyridine; Im, imidazole; NMR, nuclear magnetic resonance; ppm, parts per million; ESR, electron spin resonance. 0006-291X/88 1319

$1.50

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form res~ed.

Vol. 151, No. 3, 1988

native

Mb or Hb and why

feature (6)

it is s t a b i l i z e d

of h e m o p r o t e i n

Zn-

(7)

challenged

and

affords

oxidation

problem

and

found

here

the N i ( I I ) - P o +" w h i c h

N i - H R P . P o +'.

occurs

at e i t h e r

It has b e e n the m e t a l

Po or N i ( I I ) - P o +', whether oxidation replaced

and

HRP

oxidation

of N i - M b

relatively

unstable

shown

oxidation site

depending or not

-Hb and

-HRP

Mg-

We

have

nickel(II)-substituted

that

or p o r p h y r i n

for

(8).

that are

different

the case

using

liganded

of N i ( I I ) - M b ,

Mb

also

This

by

respectively,

it is a x i a l l y

in HRP.

has b e e n

Ru-substituted

this

hemoproteins

with

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

as c o m p a r e d

of

Ni(II)-Po

to p r o d u c e

Ni(III)-

on t e m p e r a t u r e

(9).

We have

in w h i c h

Ni-Hb

and

studied

the heme

iron

on here is

by Ni(II).

MATERIALS

AND M E T H O D S

M y o g l o b i n and h o r s e r a d i s h p e r o x i d a s e w e r e p u r c h a s e d from Sigma C h e m i c a l Co. and Toyobo, r e s p e c t i v e l y . H u m a n a d u l t h e m o g l o b i n was p r e p a r e d in the usual m a n n e r from fresh w h o l e b l o o d cell o b t a i n e d from a normal individual. Nickel-porphyrin complexes were s y n t h e s i z e d by the m e t h o d of S h i b a y a m a et al (10). Incorporation of Ni(II)-protoporphyrin (PP) or -2,4-diacetyldeuteroporphyrin (DiAcP) into a p o M b (sperm w h a l e and horse heart), apoHb (human adult) and apoHRP (isoenzyme c) was made by following the l i t e r a t u r e m e t h o d (10,12). Oxidation of n i c k e l - s u b s t i t u t e d h e m o p r o t e i n was p e r f o r m e d by adding a s t o i c h i o m e t r i c a m o u n t of i r i d a t e s o l u t i o n (K~IrCI 6 in 0.01 M HCI) to p r o t e i n s o l u t i o n (0.1 M p h o s p h a t e b u f f e r 7 pH 7.5). C h e m i c a l o x i d a t i o n of n i c k e l - p o r p h y r i n c o m p l e x e s was c a r r i e d out by adding a small a m o u n t of I~ and A g P F 6 to dichloromethane z solution. Silver hexafluorophosphate (AgPF 6) was used as a s a t u r a t e d a c e t o n i t r i l e solution. Optical measurements were c a r r i e d out using Hitachi 330 spectrophotometer. P r o t o n NMR and ESR s p e c t r a w e r e r e c o r d e d with Nicolet NT-300 spectrometer and JEOL PE-2A spectrometer, respectively.

RESULTS When

AND DISCUSSION

visible with

isosbestic

to

oxidized

(Fig.

spectrum

giving bands

was

Ni(II)-HRP

IA) was

points

a N I ( I I ) - P o +" type (Fig.

the

2B).

one

A newly at

578

with

iridate

changed

at 360,

438,

into 514,

spectrum

having

appeared

peak

nm

for

1320

(K2IrCI6),

well

the

its

UV-

broadened

one

580 nm,

broadened

eventually featureless

at 620nm m a y established

correspond Ni(II)-

Vol. 151, No. 3, 1988

A

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

g=2.001

B

395 3250 + 250 G

W 0 Z

< ~n FK

I 560

0

m <

5

~60

I

I

I

I

I

1

350

400

450

500

550

600

650

700

WAVE LENGTH (nm) Figure I. HRP upon

The UV-visible spectral changes for addition of iridate. An aliquot of

nickel-substituted saturated iridate

solution (each 5 UI) in 0 . 0 1 M HCI was added to 5 uM nickel(II)HRP (0.1 M Tris-HCI, pH 7.5), and the spectrum was measured soon after oxidation. The ESR spectrum of oxidized nickel(II)-HRP at 77 K is given in the inset.

octaethylporphyrin radical upon

spectrum

addition

~-cation was

This

sharp

spectrum

disappeared

oxidized

upon

that N i ( I I ) - P o +" Ni(II)-Mb visible

reversibly

of a r e d u c i n g

F e ( C I 0 4 ) 2. ESR

is f o r m e d

spectral

afforded

a single

When

we e x a m i n e d

spectrum

These

results

quite

of this

oxidized

1321

of

a single

77 K,

What

which show

happened

with

to

the

5.5 G

N i ( I I ) - P o +"

for UV-

case

of

at

77K

Ni(II)-Mb line

of N i ( I I ) - D i A c P - s u b s t i t u t e d

characteristic

and

oxidation-induced

similar

at g=2.001

NaBH 4

afforded at

one

unambiguously

(6-8).

2A, w h e r e

appear

peak

oxidation

of N i ( I I ) - H R P

This

unoxidized

such as N a 2 S 2 0 4 ,

in N i ( I I ) - H R P

spectrum

to the

5.5 G line width)

in Fig.

changes

The E S R

also

UV-visible

product

reduction.

( N i ( I I ) - O E P . P o + ' ) . 11

changed

reagent

(g=2.001,

is i l l u s t r a t e d

Ni(II)-HRP.

radical

width. Mb,

was

the also

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

A

N,I 0EP

:3971

o

2.5

','2

~/~422

0 z

m<1.5 0 ml <

(.2 Z

<

8~ 0£ 0 (8

5 0

350

400

450

500

550

•

600

650

701

~574

<

4:.32

388

300

T

r

I

r

I

T

I

358

488

458

S88

550

688

658

788

WAVE LENGTH ( ~ m ) Figure

2.

UV-Visible

spectral

changes

(at

room

temperature)

for

Ni(II)-Mb upon oxidation by iridate, 0.1 M Tris-HCl, pH 7.5. In the inset is shown the UV-visible spectra of Ni-OEP derivatives in dichloromethane at room temperature. Ni(II)-OEP (---), Ni(II)OEP.Po +" oxidized with I^/ and AgPF 6 (-----), Ni(III)-OEP(Py) 2 ( - - ) oxidized with 12 and AgPF 6 in the presence of pyridine.

obtained

and

Replacement substantial porphyrin

ESR spectrum of

2,4-vinyl

complexes:

CH2CI 2.

This

changes

of Po +',

porphyrin,

g=2.001

formed

changed

reversibly in c o n t r a s t

that

to

to a l m o s t

H R P . P o +'.

N i ( I I ) - P o +"

Uv-visible

spectrum

formed

returned

g=2.004.

groups

caused

model

Ni(II)

for N i ( I I ) - P P . P o +" and g=2.002 with

12 and

the z-electronic

the radical

reagent

back

at

as seen for the

in the protein.

of the reducing

line

acetyl

upon oxidation

suggesting not

a single by

g shift may reflect

addition

yield,

groups

shift of the g value,

N i ( I I ) - D i A c P . P o +"

the

exhibited

center

AgPF 6

is located

It is to be noted

unoxidized

complete

in M b w a s in t i m e

1322

spectra

reversibility not back

that

in for

so s t a b l e to

in

structural

the Po +" type UV-visible

the

for

the

in

upon

spectra a

50

%

Ni(II)that

its

Ni(II)-Mb

Vol. 151, No. 3, 1988

spectrum

within

decreased

in

exhibit

30 m i n

its

on the

As Fig. peaks,

at

been

and

of Val ppm

E11

from H20

422 nm band

398 nm band band.

by the group

while

(Fig.

of p r e d o m i n a n t

four-coordinate

It

is thus

the

four-coordinate

IA),

the

90% r e c o v e r e d likely

the

with

and

upon

the

pocket

that

(13). each

proton

with

of o x i d a n t

422 nm band

NMR

-8.4

ppm

-7.2

ppm

to

Ni(II)-

compared

was

with

the

397 nm

not

recovered.

is m i s s i n g ,

suggestive

form h a v i n g

the

395 nm b a n d

of the o x i d i z e d

N i ( I I ) - P o +" is m o r e

form of N i ( I I ) - M b

four

of

the heme:

preferentially,

reduction

which

respectively

shifted

two

proximal

to

to be c o m p a r e d

422 nm b a n d

into

nm,

of N i ( I I ) - M b . Po +" r e p r o d u c e d

For N i ( I I ) - H R P

almost

form

above

addition

was d e c r e a s e d

not

Similar

is split

in the h e m e

located

did

Imax=397

ring-current

successive

in a 50 % yield,

at

to Ni(II),

embedded

was

for N i ( I I ) - H b .

to Ni(II)

unbound

Reduction

was

of N i ( I I ) - M b

the o t h e r

signal

reduction.

band

for N i ( I I ) - M b

With

upon

ESR

spectrum

obtained

bound

y-methyl

the

five-coordinate

is p r o p e r l y

for F e ( I I ) - M b C O . the

His

the

were

nm and

as c o n f i r m e d

-6.9

Mb,

Soret

to the

form with

conformer,

Ni(II)-Mb

imidazole

Ni(II)-porphyrin

signal

the

Imax=422

(His)

coordinate

Thereafter

toward

attributed

histidine

concomitantly

Po +" f o r m a t i o n

2 shows,

one

and

intensity.

reversibility

results

have

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

than

favorably in its

which

species. formed

in

five-coordinate

form. The sharp HRP

present

finding

contrast

to Mg-

in w h i c h

changes

oxidation

with

preferential

Ni(II)-Mb, different

(6),

m e t a l l o - P o +"

spectral

of

of N i ( I I ) - P o +" in b o t h

were

iridate.

Zn-

is f o r m e d

encountered Taking

Po +" f o r m a t i o n

a protein between

(7),

conformation

four-

and

(8) and F e - M b

only

in HRP.

for Mg-,

into for

Ru-

account the

which

and

No

be

forms

in -

UV-visible

the p r e s e n t

could

is

(4) and

Zn-Mb

four-coordinate

five-coordinate 1.323

Mb and HRP

(7)

upon

results form

of

substantially of

Ni(II)-Mb

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

may be responsible for stabilization of radical center formed the porphyrin. is

In five-coordinate Ni(II)-Mb,

the radical

presumably transferred from the porphyrin to a

acid

residue,

four

resulting in degradation of the protein.

or five-coordinate

Ni(II)-PP

center

nearby

possible to prepare the reconstituted Ni(II)-Mb with

When

amino It

apoMb

an

the four-coordinate

was

the

reconstitution

obtained.

On

other

with and excess Ni(II)-PP afforded

to

equimolar

amount of Ni(II)-PP was added to apoMb, predominantly

was

predominant

form by changing the ratio of

upon the reconstitution reaction.

in

form

hand,

the

predominantly

the five-coordinate Ni(II)-Mb which did not give the porphyrin ~cation radical. In

connection

hemoproteins, of

to

the

oxidation

Ni(II)-substituted

we have also examined metal or porphyrin

the model Ni(II)-Po complexes

PP.Po.

of

such as Ni(II)-OEP and

In the absence of coordinating

they formed Ni(II)-Po +"

(Fig.

oxidation

2B).

Ni(II)-

ligand in CH2CI 2 solution,

However,

they exhibited metal

oxidation to form Ni(III)-Po in the presence of pyridine imidazole

(Im) derivatives,

visible and ESR spectra gu=2.030

(AN =16G) ,

Imax=386, products such

as confirmed by

(AN±=13G) ;

523, 558 rim, g =2.140, of

Ni(II)-hemoproteins

Ni(III)-Po

type

spectra,

g =2.289

to

for

in

(14).

accord

517,

UV-

552 rim,

The

with

oxidized

not

exhibit

the

axially

It is thus likely that

protein conformation caused by unligation of the proximal the heme central Ni(II)

is responsible

Ni(II)-Po +" in these Ni(II)-hemoproteins. protein

conformation

is

flexible

for

enough

upon

oxidation.

We tried to

1324

test

His

stabilization

In other words,

coordinated Ni(II)-Po in Mb and Hb, Ni(III)-Po formed

or

Ni(III)PPDME(Im) 2

examined here did

unliganded form of Ni-Po in hemoproteins. a

characteristic

: Ni(III)OEP(Py) 2 Imax=392,

g~=2.181

(py)

to

make

a

if the hexa-

is expected to this

by

of

be

oxidizing

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ni(II)-Mb

in

the

presence of a small

imidazole

which could bind to the heme sixth-coordination

but we did not get Ni(III) On

amount

of

pyridine

the basis of the present results,

it is tempting for us

in the heme vicinity

an important role in stabilizing the metallo-Po +" and the oxidizing equivalent of compound I for HRP and of the ES CCP

site,

species due to protein denaturaion.

propose that the protein conformation

for

or

is located in the porphyrin or

protein

to

plays second complex

depending

the

protein conformation.

It is also likely that compound I could be

basically

Mb,

formed

in

which could

be

tested

by

protein-

engineered Mb derivatives with amino acid substitution.

REFERENCES I.

2. 3.

4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

Hawson, W. D. and Hager, L. P. (1979) In The Porphyrins (Dolphin, D . e d . ) Vol. VII, pp. 295-332. Academic Press, New York. White, R. E. and Coon, M. J.(1980) Annu. Rev. Biochem. 49, 315-356. Ullrich, V.; Castle, L.; Haurand, W. (1982) In Oxygenase and Oxygen Metabolism (Nozaki, M. et al. eds.) pp.497-509. Academic Press, New York. Aviam, I.; Witterberg, B. A.; Witterberg, J. B. (1978) J. Biol. Chem. 253, 5685-5689. Poulos, T. L. and Kraut, J. (1980) J. Biol. Chem. 255, 81998205 and references cited therein. Kuwahara, Y.; Tamura, M.; Yamazaki, I. (1982) J. Biol. Chem. 1982, 257, 11517-11522. Kaneko, Y.; Tamura, M.; Yamazaki, I. (1980) Biochemistry 19, 5795-5799. Morishima, I.; Shiro, Y.; Nakajima, K. Biochemistry 1986, 25, 3576-3584. Wolberg, A. and Manassen, J. (1970) Inorg. Chem. 9, 23652367.; Dolphin, D.; Niem, T.; Felton, R. H.; Fujita, I. (1975) J. Am. Chem. Soc. 9_~7, 5288-5290.; Chang, D.; Malinski, T.; Ulman, A.; Kadish, K. M. (1984) Inorg. Chem. 1984, 23, 817-824. Shibayama, N.; Morimoto, H.; Miyazaki, G. (1986) J. Mol. Biol. 192, 323-329. Fuhrhop, J. -H. and Mauzerall, D. (1969) J. Am. Chem. Soc. 91, 4174-4181. Yamada, H.; Makino, R.; Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353. Shellnut, J. A.; Alston, K.; Ho, Jui-Y.; Yu, Nai-T.; Yamamoto, T.; Rifkind, J. M. (1986) B i o c h e m i s t r y 25, 620-627. Morishima, I.; Takatera, K.; Takeda, M. submitted for publication.

1325