Heme-Hydrogen Peroxide Complex Formation as Studied by ESR and Optical Spectroscopy

L.I. Simdndi (Editor), Dioxygen Activation and Homogeneous Catalytic Oxidation 0 1991 Elsevier Science Publishers B.V., Amsterdam

305

Heme-Hydrogen Peroxide Complex Formation as Studied by ESR and Optical Spectroscopy

Kunihiko TAJIMA*, Masato SHIGEMATSU, Junichi JINNOa, Kazuhiko ISHIZU and Hiroaki OHYA-NISHIGUCHIb

Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790, Japan. aResearch Institute of Ohtsuka Pharmeceutical C o . Ltd., Tokushima 771-01, Japan. bDepartment of Chemistry , Faculty of Science, Kyoto University, Sakyo-ku Kyoto 606, Japan. summary

Simultaneous ESR and optical measurements were carried out for the following two kind of reaction systems: 1) reactions between Fe(I1)OEP(pyridine)-O2 complex and ascorbic acid sodium salt (AsNa), and 2) reaction between Fe(II1)OEPCl and hydrogen peroxide under aklaline condition. The identical Fe(II1)OEPhydrogen peroxide complex was detected in these reaction systems. Introduction

One of the most important functions of heme enzymes have been well recognized to be the metabolisms of oxygen and peroxides in the biological systems. catalyze

Cytochrome P-450 (I), for example,

the mono-oxygenation of a wide variety of

substrates

306

by utilizing molecular oxygen coupled with NADPH dependent reductase, so called P-450 reductase. The P-450 was frequently called as the mixed

function oxidase, because, this enzyme

catalyze or at least promote the mono-oxygenation reacting with peroxides.

In addition, the catalases (2) and peroxidases

(3)

efficiently decompose hydrogen peroxide in order to protect biological tissue from the serious damages caused by peroxides. The formar catalases never oxidize substrate molecules, but the latter peroxidazes exhibit oxidation activity towards substrate molecules, in the processes of hydrogen peroxide decomposition. In the early

reaction

stages of these

heme

enzymes,

formation of heme-hydrogen peroxide complexes have frequently been assumed. The coordination and electronic structures of the hydrogen peroxide complex was still equivocal, because these complexes were very short life time and easily changed to high valent iron complexes. Recently we (4) have reported that the rapid-mixing-and-freezing method, combined

with simultaneous

ESR and optical measurements, was useful procedure to detect heme-peroxide complexes. Here we will report the mechanisms for formation of heme-peroxide complexes, based

on the results

obtained from the simultaneous ESR and optical measurements (5).

Experimental The free base of octaethylporphyrin (OEPH2) obtained from Aldrich was purified by column chromatography. The Fe(II1)OEPCl was

prepared by usual methods.

An aqueous hydrogen peroxide

solution obtained from Wako Pure Chemicals

was diluted and

supplied for measurements after calibration of cocentration by iodomery.

An ascorbic acid sodium salt

addition of equimolar

was obtained by

sodium bicarbonate to the aqueous

solution of ascorbic acid.

307

Optical spectra of the frozen slution was recorded by using a MCPD-100 spectrometer of Ohtsuka Electrinic Co. Ltd.

ESR

spectra were observed by using a JEOL FEZXG X-band spectrometer operating with lOkHz field modulation. Results and discussion

The optical absorption spectrum recorded for the frozen N,Ndimethylformamid

(DMF) solution of Fe(I1)OEP(pyridine)-O2

(l.OmM, 0.4ml) showed absorption maxima at 5 3 6 and 568 nm (Fig.

l-a)f which agreed well with that of recorded at -40 OC, as summarized in Table 1. On the ther hand, ESR spectrum recorded for the same frozen solution (Fig. 2-a) was almost silent, besides, a small amount of remaining ferric high-spin species

1.0.1 450 Fig.

500 1;

.

.

600

700

nm

Optical absorption spectra recorded at 77 K before

addition of AsNa

(a) and after addition of AsNa (b). Aqueous

ascorbic acid sodium salt (0.2Mf 0.02ml) was rapidly added to the cooled DMF solution of Fe(II)0EP(pyridine)-O2 0.4ml).

(l.OmM,

308

at g=6 and

2. Then the frozen solution was thawed at - 4 0 ° C

and

aqueous AsNa (0.2MI 0.02 ml) was rapidly added and immediately frozen at 77 K. As shown in Fig. 2-b, the E S R spectrum revealed formation of the ferric low-spin species (S=1/2) (denoted as complex A ; radical

g1=2.286, g2=2.171 and g3=1.953) with the free

(g=2.006)

species

derived

from ascorbic

acid.

The

li-TCNQ

-A

2 2 8 2.17 1.95

Fig.

2;

ESR

spectra recorde at 77 K

solution supplied

for the same frozen

for the optical measurements.

(a) E S R

observed for frozen DMF solution of Fe(II)OEP(pyridine) -02, (b) for the frozen solution prepared by

E S R spectrum observed

mixing AsNa. ESR signal of complex ( A ) was never detected by mixing the oxygen complex with AsNa above

OnC.

309

broadened ESR signal appeared in the low-magnetic field was ascribable to be the ferric high-spin species. The optical absorption spectrum recorded for the frozen solution gave a pair of absorption maxima at 556 and 604 nm, as shown in Fig. 1b). The ESR signal of complex (A) was never detected by mixing Fe(II)OEP(pyridinr)-02 AsNa,

complex with ascorbic acid instead of

but was safely recorded after addition of aqueous

solution of sodium bicarbonate.

This indicates that complex (A)

is generated by reduction of the Fe(II)0EP(pyridine)-O2 complex under alkaline condition. We have tried to generate complex (A) by using other reductants, such as NADH and NADPH, but well defined E S R signal of complex

(A) was never detected. In

addition, several kinds of aprotic solvents were used in order to stabilize the complex (A), but we found that DMF is the unique solvent to generate complex

(A) in our

reaction

condition. By the similar rapid-mixing-and-freezig method, reaction between Fe(II1)OEPCl and hydrogen peroxide was monitored under the alkaline condition. To the pre-cooled (-40°C) DMF solution of Fe(II1)OEPCl

(l.OmM,

0.4ml), an aqueous hydrogen peroxide

solution (0.2M, 0.02ml) was added in the presence of aqueou KOH (0.2M, 0.02ml). ESR and optical spectra due to the complex (A) were clearly recorded, and those spectroscopic parameters agreed with each other within the experimental error, as summarized in Table 1. In addition, formation of complex (A) was also recognized by addition of AsNa to the Fe(II1)OEPCl under the aerobic condition. In this case,

however, presence

of pyridine was indespensable to detect complex (A) (Table 1). As summarized in Table 1, the ESR and optical parameters of the complex (A) showed excellent agreenment with those of the complex Fe(III)TPP(-OH) (-OOH) (g1=2.269, g2=2.162 and g3=1.961, absorption maxima 562 and 604 nm) (6). From comparison

of

the

310

observed optical parameters of complex hydrogen peroxide complex,

(A) and

Fe( 111)TPP-

(A) is assumed to be the six

coordinate Fe(II1)OEP-hydrogen peroxide complex, in which the axially ligating pyridine of the Fe(II)0EP(pyridine)-O2 will be replaced with the hydroxide anion generated under condition.

As illustrated in Scheme 1, an identical complex

Fe(III)OEP(-OH)(-OOH) oxygen

alkaline

complex,

is generated by

or by

mixing

reduction of Fe(I1)OEP

hydrogen

peroxide

to

the

Fe(III)OEPCl, under alkaline condition. In addition complex (A) was also generated by reduction of Fe(II1)OEPCl with AsNa under oxygen condition. These observeation results demonstrated

that

oxygen molecule binding at the axial position of heme is activated to -0OH

anion reacting with reductants. The complex

(A) will be the practical model complex f o r the one-electron reduced species of P-450 oxygen complex (1). Table 1 Optical and ESR spectroscopic parameters observed for

Fe(II1)OEP-hydrogen peroxide complex and relating complexes. comp1ex

absorption max

Fe ( 11)OEP (Py) O2

g1

g2

g3

ref.

536 568 405 536 563

II

7

* *

Fe ( 111)OEP ( -OH) (-OOH) a

556 604

2.286 2.171 1.953

Fe ( 111)OEP (-OH) (-OOH)

556 604

2.287 2.171 1.955

Fe ( 111)OEP ( -OH)

( -0OH)

557 606

2.287 2.170 1.949

*

Fe (111)TPP (-OH)(-OOH)

562 604

2.269 2.162 1.961

6

____________________-------------------------------------

*, +

present work, a)Fe(II)OEP(Py)O, KOH, c)Fe(III)OEPCl

+AsNa, b)Fe(III)OEPCl + H202

+ AsNa + Py +

02.

311

I OX 00 IY h

n

H

Scheme

1,

Probable

mechanisms

for

the

six

coordinate

Fe(II1)OEP-hydrogen peroxide complex formation. References

R. E. White and M. J. Coon, Ann. Rev. Biochem., 49 (1980) 315 L. P. Hager, D. L. Doubek, R. M. Silverstein, J. H. Hargis and J. V. Martin, J. Am. Chem. SOC., 94 (1972) 4364. G . R. Schonbaum and B. Chence, in P. D. Boyer (ed.) The Enzymes Vol. 8C, Academic Press, New York, 3rd edn., 1973, pp. 363 K. Tajima, J. Jinno, K. Ishizu, H. Sakurai and H. OhyaNishiguchi, Inorg. Chem., 28 (1989) 709; K. Talima, Inorg. Chim. Acta, 169 (1990) 211 K. Tajima, M. Shigematsu, J. Jinno, K. Ishizu and H. OhyaNishiguchi, J. Chem. Soc., Chem. Commun., (1990) 144 K. Tajima, InOrg. Chim. ACta, 163 (1989) 115 C. H. Welbone, D. Dolphine and B. R. James, J. Am. Chem. S O C . , ~ (1981) 2869