generation of phenoxy radicals by methemoglobin-hydrogen peroxide studied by electron paramagnetic...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 164, 540-547 (1973)

Generation of Phenoxy Radicals by Methemoglobin-Hydrogen

Peroxide Studied by Electron Paramagnetic Resonance

TAKESHI SHIGA AND KAZUHIKO IMAIZUMI

Department of Physicochemical Physiology, Medical School Osaka University, 33, Joancho Kita-ku, Osaka, Japan

Received August 23, 1972

The free radical intermediates of phenol derivatives, produced by the methemoglobin-hydrogen peroxide system at pH 5 and 7, are detected by electron paramagnetic resonance equipped with a continuous-flow apparatus. All the radicals from phenols are the phenoxy radicals, as identified by analyzing the observed hyperfine structures of the spectra with the aid of SCF-LCAO MO calculations. Comparing with the reaction of Fenton’s reagent, it is concluded that free OH radical, even if it exists, is not liberated into the solution in the methemoglobin-hydrogen peroxide system.

One-electron oxidation of the organic substrates catalyzed by peroxidase has been established (I), and recognized to be an important step of biological oxidation of phenols (2). Although the studies on the final products of various biological and chem- ical oxidation systems have been made (3-5), not much attention has been paid to the intermediate radicals. Using electron paramagnetic resonance (EPR), we have been accumulating the knowledge of organic intermediate radicals produced by Fent,on’s reagent (6-lo), which are formed either by hydrogen abstraction or by hydroxylation of various substrates. As an extension of these studies, we have studied the “peroxidation” reaction of phenol derivatives catalyzed by methemoglobin (YIetHb)-H,02 system.

Peroxidative reaction of metmyoglobin and methemoglobin has been extensively studied (II), compared with peroxidase and catalase. The kinetic features of the reac- tions differ from each other, but the ‘lperoxi- dation” commonly occurs. Among the hema- tin proteins, RletHb has certain advantages as an experimental material, compared with peroxidases, because (1) a large quantity is available and (2) the t,ertiary st,ructure is known. Therefore, we have first identified

the free-radical intermediates generated by MetHb-HzOz system and compared them with the radicals produced by Fenton’s reagent (Fe(I1) + H,Oz).

This paper, thus, is primarily concerned with the identification of the organic free radicals generated by MetHb-Hz02 system, but the kinetic feature of the int’ermediate will be described separately.

MATERIALS AND METHODS

Human hemoglobin was prepared by the method of Drabkin (12) from out-of-date bank blood. The conversion of oxyhemoglobin to MetHb was carried out by adding a minimum quantity of ferricyanide (13). Then the solution of MetHb was dialyzed. The organic materials and the reagents were of reagent grade, and used without further purification.

A Varian E-12 EPR spectrometer equipped with a continuous-flow system was used. The de- sign and the characteristics of the flow system have been described elsewhere (6, 14). A solution of MetHb (ca. 0.2 mM) and a solution containing Hz02 (50 mM) and phenol derivatives (usually 10 mM, depending on the solubility) were mixed just before the EPR cavity. The dissolved oxygen in the solution was removed by bubbling the nitrogen gas or by stirring under the stream of nitrogen gas (99.999yG). Without phenols, an unresolved broad EPR signal arising from MetHb

540

Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

PHENOXY RADICALS IN

itself appeared (15), similarly to the reaction between metmyoglobin and Hz02 (16-20). In the presence of phenols, however, the MetHb radical was replaced by a well-resolved signal of phenol radical which we shall describe later. All the EPR spectra were recorded at a flow rate of 0.2- 0.4 liter/scan (= 4 min), which corresponds to the spectra 10-15 msec after mixing the solutions. Fremy’s salt was used as a standard. All the ex- periments were carried out at 24°C.

A Hitachi Hitac 10 digital computer was employed for theoretical calculations.

R,ESULTS

The EPR spectra of the radicals from phenol and p-Cl-phenol, generated by RletHb-HzOz syst,em at pH 5.0, are shown in Fig. 1. The same spectra are detectable at pH 7.0, but no EPR signal is observed at pH 8.3. The hyperfine structures a.re easily analyzed and their coupling constants agree with the reported values of the corresponding phenoxy-radicals (21), which are produced by oxidation of phenols wit,11 ceric ion at unknown pH.

In Fig. 2, EPR spectra of the radicals from p-, m-, and o-hydroxybenzoates, produced by MetHb-HzOn at pH 5.0, ale shown. The hyperfine coupling constants

METHB-Hz02 REACTION 541

also agree with the reported values of the corresponding phenoxy radicals (21).

The EPR spectra of the radicals from p-, m-, and o-methoxy phenols, generated by MetHb-HZOz at pH 5.0, are shown in Fig. 3. Except for m-derivative, the observed hyperfine structures can be analyzed, and the molecular structures of the radicals can be identified as the phenoxy radicals, by comparing with the other spectra. However. a small proton coupling cannot be assigned t.o one of two ?n-prot.on of o-methoxy- phenoxy radical.

In Fig. 4, EPR spectra of the radicals from p-, nz-, and o-cresols, produced by MetHb- H202 at pH 5.0, are shown. The radicals are identified as the phenoxy radicals, except for o-cresol radical of which the spec- trum cannot be uniquely analyzed. The phenoxy radical from p-cresol is the same as one observed in the horse radish peroxidase reaction (22).

For comparison, the oxidation of phenol and p-hydroxybenzoate by Fenton’s reagent at various pH values is studied. As demonstrated in Fig. 5, the radical species from phenol are highly dependent on pH of the solution: (a) at pH 1 (in 0.1 K HzS04 ,

, 5 gauss

Cl

,.--.

0

' : 1 I '._'

0.

I I

FIO. 1. EPR spectra of phenoxy radicals of phenol and p-cl-phenol, produced by MetHb-HzOz. Conditions: microwave power 10 mW; modulation 100 kc, 0.5 G; time constant 1.0 set; scanning speed 40 G/‘4 min for phenol and the same but modulation amplitude of 0.63 G for p-cl-phenol radicals.

542 SHIGA AND IMAIZUMI

FIG. 2. EPR spectra of phenoxy radicals of p-, m-, and o-hydroxybenzoates, produced by MetHb- H202. Conditions: same as in Fig. 1, modulation smplit,ude of 0.63 G for p- and o- and 0.5 G for m derivatives.

5 mM FeSOl , and 20 m H,O,), a phenoxy radical is observed (Fig. 5a), (b) at pH 5.0 (in 0.1 M acetate buffer, 5 rnM FeS04 + 5 rnM EDTA and 20 mM HzOz), an o-hydroxycyclohexadienyl radical (23) is recognized; in addition, at least one isomer may exist (Fig. 5b), (c) at pH 7.0 (in 0.1 WI phosphate buffer, 5 m&I FeS04 + 5 mM EDTA and 20 mM H202), p-semiquinone and o-semiquinone are observed together (Fig. 5c), and (d) at pH 9.0 (in 0.1 M pyrophosphate buffer, 5 rnM FeS04 , and 20 mM HzOz), only o-semiquinone is detected.

We have observed similar pH-dependent phenomena with the radicals from p-hydroxybenzoate: at pH 1 phenoxyradical is detected, at pH 5.0 o-hydroxycyclohexadienyl radical is recognized and at pH 9.0 o-semiquinone is observed.

DISCUSSION

Spin Densities and Hype&e Coupling Con- stants

In order to confirm the above identification and to assign the hyperfine couplings to

PHENOXY RADICALS IN METHB-Hz01 REACTION 543

OCH,

m-methoxyphenol

OCH,

FIG. 3. EPR spectra of phenoxy radicals of p-, m-, and o-methoxyphenols, produced by MetHb-HzOz. Conditions: same as in Fig. 1, modulation amplitude of 0.25 G for p- and o- and 0.8 G (scanning speed 100 G/4 min) for m derivatives.

the molecular structures, the LCAO-MO calculations with McLachlan modification (24) are carried out employing the Hiickel parameters listed in Table I. The results are summarized in Table II. The observed hyperfine coupling constants (A) are plotted against the calculated spin densities (p), as shown in Fig. 6, they agree well. A least- square fitting to the McConnell’s equation

(25)

A = Qep

gives the Q-value of -27 G, which is a reasonable value (26).

These phenoxy radicals are definitely free in solution and not bound to MetHb, since (1) each hyperfine line is sharp and averaged well, and (2) the observed coupling constants are well predicted by the theoretical

calculations (i.e., no distortion of the spin density distribution due to complexing).

Comparisons with Other Oxidation Systems

The pH-dependent variation of the intermediate free radical, observed with Fenton’s reagent, have been tentatively explained as follows (23,27) : the hydroxy cyclohexadienyl radical, which is the initial product by OH- radical attack (e.g., in Ti(II1) + H,O, system), tends to undergo (l), at lower pH, an acid-catalyzed reaction giving phenoxy radical or (2), at higher pH, a base-catalyzed reaction giving semiquinone. If the nature of the reactivespeciesinvolved in MetHb-HzOz system is an OH radical as proposed by George (11,2S), the hydroxy cyclohexadienyl radicals could be detected, since the radicals are stable enough in order to be observed by the present technique at pH 5.0. However,


no hydroxy cyclohexadienyl radical is observed in MetHb-H202 system. Further, the

the hydroxycyclohexadienyl radicals change

radicals produced by MetJIb-HzOz system to the semiquinones at pH 7 as demonstrated in Fig. 5. Therefore, free OH radical is def-

at pH 7.0 are the phenoxyradicals, whereas initely not liberated into the solution in

--. 0 : ; .--’

0. v

I I IO gauss

FIG. 4. EPR spectra of phenoxy radicals of p-, m-, and o-cresols, produced by MetHb-HIOz. Condi- tions: same as in Fig. 1, modulation amplitude of 0.5 G for m- and 0.32 G for o-cresol; scanning speed 100 G/2 min, time constant 0.3 set, modulation amplitude 0.63 G for p-cresol.

TABLE I H~CKEL PARAMETERSO

. C-

(1 .4)2oo

c- -

. ~0.7!,~,(a.5)_,~, H3

C-

(O.Wlc; .

c (1.2) e”

c - o . ,(1-G)’ ’

1.6

C- -c-

b7)2~ob.7)_, I2.5)-o 2 H3

. .

a The numbers in parentheses are bond integrals, and the others are Coulomb integrals. Mc- Lachlan parameter, A, is 1.2. These parameters are the same as employed in Ref. 10, for calculating the spin densities of the hydroxycyclohexadienyl radicals of benzoate derivatives.

PHENOXY RADICALS IN METHB-Hz02 REACTION 545

b) PH 5

I I

I I I II I

d) P” 9

FIG. 5. EPR specka of radicals of phenol, produced by Fenton’s reagent at various pH values. Con- ditions: same as in Fig. 1, but modulation amplitude of 0.63 G throughout; scanning speed of 100 G/4 min for b. The arrow indicates 5 G for a, c, d and 7.5 G for b.

FIG. 6. The relation between the calculated spin density and the observed hyperfine coupling const.ant.

MetHb-HzOz system. If OH radical is in- volved in MetHb-HzOs system, the initial products, formed by the reaction with OH radical, must change to phenoxy radicals before they are liberated from t,he heme pocket into the solution.

Concerning peroxidase reaction, only few aromatic intermediate radicals have been observed (1, 22, 29). For example, p-methyl- phenoxy radical has been detected in horse radish peroxidase reaction at pH 6.0 (22). We are also able to detect the phenoxy radicals from p-cresol, phenol, p-hydroxybenzoate, and o-methoxyphenol in horse radish peroxidase reaction at pH 5.0. If such phenoxy radical formation is a common phenomenon in hematJin-enzyme systems, the liberation of the phenoxy radicals, in- dependently of pH, may bc an import,ant


TABLE II HYPPRFINE COUPLING CONSTANTS (OBSERVED) AND SPIN DENSITIES (CALCULATED)’

Substituents Positions

2 3 4 5 6

-

4-Cl

-i-coo-

5-coo-

B-COO-

4-CH3

5-CH3

4-OCHa

6-OCHa

(-Z4) 10.01 (0.360) (-Z4)

6.50 (0.221)

6.3s (0.135)

6.35 (0.232)

(::;;4)

-

(E5) l.Sb (0.009)

Cl l.Sb (0.303) (kilk,

6.36 (0.232) ,-~:~;4,

1.90 (-0.074)

1.80 (-0.054)

(-Z4)

(&4)

(:::;8)

CHa 12.20 (0.345)

1.77 (-0.012)

(X4) (:::;8)

(%S)

4.00 (0.150)

OCHa 1.78 (0.278)

(;:zl) (-Z7) 8.88

(0.367) CH, 1.65

(-0.058)

(ZiO) (JOlO) OCHI 1.65

(0.326) (JOlO)

4.20 (0.109) (:.WS) (::.k) (-::::2)

a Hyperfine coupling constants are expressed in gauss. Numbers in parentheses are the spin densities.

b Due t,o overlapping, the approximate values are obtained.

fact for considering the biological oxidation 5. STAUDINGER, HJ., KERI~KJARTO, B., ULLRICH,

of phenols. V., AND ZUBRYCKI, Z. (1965) in Oxidases

At the present stage, we have clearly and Related Redox Systems (King, T. E.,

demonstrated the generation of the phenoxy Mason, H. S., and Morrison, M., eds.)

radicals by MetHb-H20z system and the Vol. 2, p. 815, Wiley, New York.

differences of the intermediate free radicals 6. SHIGA, T. (1965) J. Phys. Chem. 69, 38O5.

between MetHb-HzOz system and Fenton’s 7. SHIGA, T., BOUKHORS, A., AND DOUZOU, P.

reagent. Further kinetic studies are in prog- (1967) J. Phys. Chem. 71, 3559.

ress in order to estimate the phenomena 8. SHIGA, T., BOUKHORS, A., AND Douzou, P.

(1967) J. Phys. Chem. 71, 4264. occurring in the heme pocket. 9. SHIGA, T., AND ISOMOTO, A. (1969) J. Phys.

Chem. 73, 1139.

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1. YAMAZAKI, I., MASON, H. S., AND PIETTE, J. Phys. Chem. (in press).

L. H. (1960) J. Biol. Chem. 236, 2444. 11. GEORGE, P., AND IRVINE, D. H. (1956) J.

Colloid Sci. 11, 327. 2. WATERS, W. A. (1971) J. Chem. Sot. (B), 2O26. 12. DRABKIN, D. L. (1946) J. Biol. Chem. 146. 3. SEASON, H. S. (1957) Advan. Enzymol. 19, 79. 703. 4. BUHLER, D. R., AND asoN, H. S. (1961) 13. ANTONINI, E., AND BRUNORI, M. (1971) in

Arch. Biochem. Biophys. 33, 424. Hemoglobin and Myoglobin in Their Reac-

PHENOSY RADICALS IN METHB-H-02 REACTION 347

tion with Ligand, p. 41, North-Holland, Amsterdam.

14. SHIGA, T., BOUKHORS, A., AND Douzou, P. (1968) in Recent Development of Magnetic Resonance in Biological Systems (Fuji- wara, S., and Piette, L. H., eds.) p. 146, Hirokawa, Tokyo.

15. GIBSON, J. F., AND INGRAM, D. J. E. (1956) Nature (London) 178, 871.

16. GIBSON, J. F., INGRAM, D. J. E., AND NICH- OLLS, P. (1958) Nature (London) 181, 1358.

17. KING, N. K., AND WINFIELD, M. E. (1963) J. Biol. Chem. 233, 1520.

18. KING, N. K., LOONEY, F. D., AND WINFIELD, M. E. (1964) Biochim. Biophys. Acta 88, 235.

19. THEORELL, H., AND EHRENBERG, A. (1952) Arch. Biochem. Biophys. 41, 442.

20. BRILI,, A. S., EHRENBERG, A., AND DEN HAR-

TOG, H. (1960) Biochim. Biophys. Acta 40, 313.

21. STONE, T. J., AND WATERS, W. A. (1964) J. Chem. Sot., 213.

22. OHNISHI, T., YAMAZAKI, I., IYANAGI, T., NAKAMURA, T., AND YAMAZAKI, I. (1969) Biochim. Biophys. Acta 172, 357.

23. JEFCOATE, C. R. E., AND NORMAN, R. 0. C. (1968) J. Chem. Sot. (B) 48.

24. MCLACHLAN, A. D. (1960) Mol. Phys. 3, 233. 25. MCCONNELL, H. M. (1956) J. Chem. Phys.

24, 633, 764. 26. CARRINGTON, A. (1963) Quart. Rev. 17, 67. 27. JEFCOATE, C. R. E., LINDSAY-SMITH, J. R.,

AND NORMAN, R. 0. C. (1969) J. Chem. Sot. (B), 1013.

28. GEORGE, P., AND IRVINE, D. H. (1952) Bio- them. J. 62, 511.

29. PIETTE, L. H., BULOW, G., AND YAMAZAKI, I. (1964) Biochim. Biophys. Acta 88, 120.

generation of phenoxy radicals by methemoglobin-hydrogen peroxide studied by electron paramagnetic...

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