electron spin resonance study on peroxidase- and oxidase-reactions of horse radish peroxidase and...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 167, 469-479 (1975)

Electron Spin Resonance Study on Pe,roxidase- And Oxidase-

Reactions of Horse Radish Peroxidase and Methemoglobin

TAKESHI SHIGA’ AND KAZUHIKO IMAIZUMI

Department of Physicochemical Physiology, Medical School, Osaka University, 33, Joancho, Kitaku,

Osaka, Japan

Received July 9, 1974

The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction

of methemoglobin, were studied by electron spin resonance spectroscopy.

The difference between the peroxidase- and oxidase-reactions of HRP are demonstrated,

i.e., the ferro horse radish peroxidase-0, system attacks both phenols and benzoate yielding

unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish

peroxidase-H202 system reacts only with phenols and naphthols producing the phenoxy-

and naphthoxy-radicals.

Phenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H20,

and methemoglobin-H202, occurs independently of the molecular sizes of phenoIs but dependently on their redox-potentials.

On the basis of kinetic studies on methemoglobin-H,O, system, the existence of a

reactive intermediate complex between methemoglobin and H,O, is proposed, which may

be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the heme-

pocket of methemoglobin.

In order to elucidate the mechanism of heme-protein catalyzed oxidation reactions, the knowledge on the nature of the reactive species, i.e., various forms of the heme-oxygen complexes, is necessary. For example, three reactive oxidation states of peroxidases, compound-I, -II, and -III, are different in their physical properties, more- over the characteristics of the compounds differ from protein to protein. In 1957, Mason et al. have shown, by analyzing the reaction products of horse radish peroxidase (HRP)’ reaction, that the hydroxylation of benzenoid substrates occurs in the reaction of HRP-dihydroxy fumarate-

1 Present address: Department of Physiology, Med-

ical School, Ehime University, Shigenobu-cho, On-

sengun, Ehime, Japan. 2 Abbreviations used: HRP, horse radish peroxi-

dase; MetHb, methemoglobin; esr, electron spin reso-

nance; DHF, dihydroxyfumarate; and p-OH-B, p- hydroxy benzoic acid.

(DHF)-O2 system but not in the reaction of HRP-H20z system (1, 2). Also, a marked difference in the reaction rates towards organic substrates, among the HRP-compounds, has been known (3).

Electron spin resonance (esr) technique would be a powerful tool for solving the above problem, at one hand for the deter- mination of the magnetic properties of the iron atom, and on the other hand for the detection of the intermediate organic free radicals during the HRP-H,02 reaction. Yamazaki et al. have already detected the free radical of ascrobate, demonstrating the occurrence of one-electron oxidation-reduction in HRP-H20, system (4). Similar evidence has been accumulated with many hydrogen donors, i.e., DHF (5, 6), triose reductone (6)) chlorpromazine (7), lignines (8, 9), hydroquinones (5, 6, lo), p-

phenylenediamine (6, ll), p-cresol (10, 12) and p-hydroxybenzoate (12).

469

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

470 SHIGA AND IMAIZUMI

The nature of the reactive heme-oxygen complexes differs depending on their oxidation states, as well as on their heme environment. Unfortunately, the ternary structures of heme-proteins are no known except for myoglobin and hemoglobin. Therefore, we planned to compare the characters of the reactive species involved in the HRP and methemoglobin (MetHb) reactions, by observing the free radical intermediates of benzenoid substrates during the reaction of HRP-H202, MetHb- H,O, and ferroHRP-0, systems.

In the present paper, the similarity between the HRP-H,O, and MetHb-H,O, systems, the difference between the ferroHRP-O2 and HRP-H20z systems, and the kinetics of MetHb-H,O, reaction will be briefly presented, on the basis of the esr observations of short-lived, organic free radicals generated from phenols, naphthols and benzoate.

MATERIALS AND METHODS

HRP was purchased from Sigma, and further purified on a Sephadex G-25 column (for desalting) and on a CM-cellulose column (for purification). The final absorbance ratio (R.Z.) was 2.5-3.0, the activity measured by a guaiacol test was close to the reported values, and the concentration was determined spectrophotometrically using e = 9.5 x 10’ cm-’ mall’ at 403 nm (13). MetHb was prepared from out-of-date bank-blood as described previously (12). Other chem- icals were of reagent grade and were used without further purification.

FerroHRP was prepared in a modified Thumberg tube: after Na-gas (99.999%) flush, the HRP solution was anaerobically titrated by dithionite solution through a serum cap; the completion of the reduction was monitored spectrophotometrically. DHF and dithionite were dissolved in vacua using a H-shaped Thumberg tube sealed with a serum cap, then N,-gas was introduced. The solutions of the reaction constit- uents were transferred into the reservoirs of the apparatus (described below) under NZ-gas stream, except for O,-saturated buffer. 0.1 M acetate buffer, pH 5.0, was used throughout.

A Varian E-12 spectrometer was used for esr measurement. A Hitachi F-5 pH meter, a Hitachi 124 spectrophotometer and a Hitachi Hitac-IO digital computer were employed for the measurements of pH and absorption spectra and for the theoretical calculations, respectively.

A continuous flow system was used for room temperature esr experiments, of which the design and characteristics were described elsewhere (14, 15).

This apparatus assures us to observe the phenomena occurring 5-30 ms after mixing two solutions. How- ever, since our apparatus has originally been con- structed for the study on a model chemical reaction (14, 15, 16, 17, 18, 19) it has the following disadvant- ages: (1) the dead space between the reservoirs and the mixing cell is as large as half dl; (2) the dead time of few seconds is required in order to obtain a desired flow rate; and (3) a stopped flow experiment is im- possible. In any rate, a large volume of the solutions is required.

A rapid quenching apparatus was made by Union Giken Co., which consisted of a pair of lo-ml reservoir and 1.25ml syringe (derived by N,-gas pressure) and a 4-jet-mixer. The reacting solution (one shot = 0.5 ml) was directly injected into a funnel filled with liquid nitrogen, which was dipped in the liquid nitrogen dewar and in which a piece of a stainless- steel net was placed. The lumps of frozen solution was ground by a glass-rod, then the fine powder was collected into esr sample tube (i.d. = 3.5 mm) attached to the funnel and pressed down with a plastic rod (o.d. = 2 mm). The esr sample tubes were immersed in liquid nitrogen before the measurement. esr observations were carried out using a Varian E-4531 cavity and E-275 variable temperature acces- sory operated at -170°C. Once the sample tube was placed into the cavity, there was no way of sealing the top end of the sample tube, because of gas expansion. Some radicals diminished (due to auto-oxidation by air) within two hours during the esr measurement, thus the spectra were recorded as quick as possible (a fast sweep and a short time constant) without regard- ing a poor S/N ratio. The quantitative reliability of the above procedure has been tested by monitoring the esr signal of hemoglobin-NO produced by mixing the deoxyhemoglobin and NO-saturated buffer. An error of about 20% was obtained.

Fremy’s salt was used for the concentration measurements of free radicals.

RESULTS

Rapid Freezing Experiments on HRP

The esr spectra obtained with various reaction mixtures are shown in Fig. 1.

(a) ferroHRP + 0, (Fig. la). The sample should contain a certain amount of oxy-peroxidase, but no signal was observed. The result coincided with that of Blumberg et al. (20), though our experi- mental conditions (ferroHRP 0.6 mM

mixed with O,-saturated buffer, at -170°C) were far less sensitive than their conditions (ferroHRP 1.1 mM, at 15°K) in respect to (i) concentration of oxy-peroxidase, and (ii) ambient temperature.

ESR OF HRP AND MetHb REACTIONS 471

‘_j * cl d)

--

e)

p +

FIG. 1. Low temperature ESR spectra of various reaction mixture (rapid quenching experiments).

(Top) HRP(140 ~M)-H~O~ (100 mM) + p-OH-B(10 mru). Conditions:field sweep 4000 G/4 min, modulation 100 kHz, 5 G, incident microwave power 10 mW (9.09GHz); at - 170°C.

(Bottom, a-f) Various reaction mixtures; spectra around g = 2 region. (a) ferroHRP(620 ~M)-O~ (saturated); (b) ferroHRP(620 +I)--G,(saturated) + p- OH-B(10 mM); (c) HRP(98 fire) + O*(air equilibrated)-DHF(lCl mM); (d) HRP(98 FM) + 01 (air equilibrated) + p-OH-B(10 mM)-DHF(10 mM); (e) HRP(87 p~)-H,t3~(250 mM); (f) HRP(87 NM)-H,0,(250 mM) + p-OH-B(10 mM).

Conditions: field sweep 106406 G/4-8 min, modulation 100 kHz, l-2 G, incident microwave power 10 mW; at -1’70°C. (The horizontal arrow indicates 10 G, and the mark indicates the position of the center of DPPH signal.)

(b) ferroHRP + 0, and p-OH-B (Fig. lb). When p-hydroxy-benzoic acid (p- OH-B) was added to the reaction mixture, an asymmetric signal (AH = 14 G) ap- peared at g = 2.00, of which the saturation behavior against the incident microwave power was shown in Fig. 2. When p-OH-B was replaced by 2,6-dimethylphenol or by benzoic acid, a similar signal was observed. The nature of the paramagnetic species will be discussed later.

(c) HRP and 0, + DHF (Fig. lc), and (d) HRP, p-OH-B and 0, + DHF (Fig.

Id). In both conditions, a weak, asymmet-

I I I 0 5 IO 15

Jmlcrowave power (mW)

FIG. 2. Microwave power saturation of the esr signals produced by HRP-H,O? and ferroHRP-0, systems. - x -:HRP(140 n~)-H~0,(100 mM) + p-OH-B( 10 mM); 3-:ferroHRP(620 &-02 (saturated) + p-OH-B(10 mM); -&-:ferrolHRP (620 r&f-Gr(saturated) + benzoate (10 mM); Conditions: same as those of Fig. If and b.

ric signal (AH = 6 G) was detected at g = 2.00. Unfortunately, DHF contained un- known impurities, which gave a broad esr signal at high gain, thus a quantitative study was abandoned. The shape of the observed spectrum did not seem to agree with the reported spectrum of the superoxide anion (21). Therefore, the radical may originate from DHF.

(e) HRP + H20, (Fig. le). An asymmetric signal (AH = 8 G) was detected, which was already assigned to be superoxide anion by Nilson et al. (22). This signal saturates with the incident microwave power, therefore, a structure such as Fe(R)-0; (analogous to Fe@-NO (23)) would not be the case. Also, compound-I and -11 showed no esr signal (20) at 1.4”K. Without detailed kinetics, thus, it is uncer- tain whether or not the liberated superoxide anion is the main product of the reaction.

(f) HRP + HzO, and p-OH-B (Fig. If). The above O,-like signal was completely replaced by a symmetric signal (AH =‘ 6 G) at g = 2.00, which was easily saturated against the incident microwave power (Fig. 2). The signal arised undoubtedly from organic free radical, which could be observed at room temperature by means of a continuous flow method (see below).

Room Temperature Experiments on HRP

As stated in preceding section, our continuous flow apparatus requires so much


volume of HRP solution, thus it is inade- quate for enzyme kinetics. A series of quantitative experiments were, therefore, carried out, flowing some liters of HRP solution.

(a) ferroHRP + O2 without or with p- OH-B. In both cases, no signal was detected under conditions; ferroHRP (2.3 PM), O,-saturated buffer, p-OH-B (10 mM),

at pH 5.0 (0.1 M acetate buffer). The negative result is understandable, since the concentrations of paramagnetic species would be too low (see the results of rapid quenching experiments).

(b) HRP + HzOz. Again, no signal was observed, due mainly to low concentrations of radical species.

(c) HRP + H,Oz and p-OH-B (Fig. 3a): A signal showing well resolved hyperfine

a)

l IO gouss

FIG. 3. Room temperature esr spectra of phenoxy- radicals of (a) p-hydroxy-benzoate, (b) 4-methyl-, and (c) 3,5-dimethyl-phenols, produced by HRP-HnO, system. (a) HRP(0.58 PM)-HI0,(5 mM) + p- OH-B(10 mhQ; (b) HRP(2.3 PM)-H,O, (10 msr) + 4-methylphenol (10 mM) (c) HRP(2.3 PM)-H,O, (10 mM) + 3,5-dimethyl-phenol (7 mM). Conditions: field sweep 100 G/2 min, modulation 100 kHz, (a) 1.25, (b) 0.63, and (c) 0.5 G, incident microwave power 10 mW; at 17°C.

lines was detected, which arised from phenoxy-radical of p-OH-B.

(d) HRP + H,Oz and benzenoid substrates (Fig. 3b,c and Fig. 6d). Similarly, almost all phenols tested gave the corresponding phenoxy-radicals (Table I). The identification of radical structures was made by (1) a comparison of the observed spectra with those reported by Stone and Winters (24, 25) (Ce(II1) + phenols) or by ourselves (12) (MetHB + HzO, and phenols); and (2) a comparison between the analyses of hyperfine splittings and the spin densities calculated by McLachlan method (26). The observed hyperfine splitting constants agree well with the theoretical spin densities (12).

Upon addition of p-nitro- and 2,6-dini-

TABLE I

LIST OF SUBSTRATES, TESTED IN ROOM TEMPERATURE EXPERIMENTS

Compounds MetHb- FE- I-LO,” 1 1’

Phenol + + 4-Carboxy-phenol + + 3-Carboxy-phenol + 2-Carboxy-phenol + + 4-Methyl-phenol + + 3-Methyl-phenol + 2-Methyl-phenol + 4-Methoxy-phenol + + 3-Methoxy-phenol + P-Methoxy-phenol + 4-Chloro-phenol + 4-Nitro-phenol - -

3-Nitro-phenol -

2-Nitro-phenol -

2,6-Dimethyl-phenol + + 2,6-Diemthyl-phenol + + 3,5-Dimethyl-phenol + + 2,4,5-Trimethyl-phenol + + 2,3,5,6-Tetramethyl-phenol + + 2,6-Dinitro-phenol -

l-Naphthol + + 2-Naphthol + + 2-Naphthol-6-sulfonate + + 2,7-Dihydroxy-naphthalene + +

Benzoic acid -

n +, esr signal detected; -, no signal detected; and blank, not tested.


tro-phenols and benzoic acid, however, no signal was detected.

Room Tempera&r-e Experiments on MetHb

(a) MetHb + H,Oz (Fig. 4). An asymmetric signal (AH = 13 gauss) at g = 2.00 with unresolved hyperfine structures was observed, of which the intensity did not saturate against the incident microwave power. The radical was first detected by Gibson and Ingram (27) and studied kinetically by King and Winfield (28, 29). The best spectrum is shown in Fig. 4. Based on the shape of the spectrum, it is clear that this radical possesses a number of hyperfine structures and has an anisotropy in its g-values. The radical may be explained, similarly to the case of cytochrome c peroxidase (30), as to be a radical of certain residue in MetHb itself, which is in a situation of “slowly tumbling.” The signal did not saturate against the incident microwave po’wer, therefore, the radical might be closely related to the central iron atom or might be in a situation of “rapid electron exchange.” However, much elabo- rate method (e.g., ENDOR) will be needed in order to justify the nature of MetHb radical.

Changing the flow rates, the formation rates of Met.Hb radical could be determined as a function of H,Oz concentra-

FIG. 4. Room temperature spectrum of Methemo- globin radical and its microwave power saturation. MetHb(103 r~)-H,0~(25 mM). Conditions:field sweep 100 G/4 min, modulation 100 kHz, 4 G, incident microwave power 10 mW; at 17°C. (The mark indicates the position of the center of ON- -(SO,),signal.)

tions, unless the bubble formation dis- turbed the esr observation at higher concentrations of HzO,. The reaction followed Michaelis-Menten type kinetics (see Fig. 7), and K, = 40 mM and Vjheme = 40 s-l were obtained in acetate buffer (0.1 M, pH 5.0) at 17°C.

MetHb radical disappeared so slowly that we could not follow the decay process by our apparatus.

(b) MetHb + H,Oz and p-OH-B. For quantitative purposes, p-OH-B is adopted as a substrate throughout, since (1) the signal of p-OH-B phenoxy-radical (12, 24, 25) consists of 9 distinct hyperfine lines, of which the intensity ratios are 1:2:1:2:4:2:1:2:1 due to two sets of two protons, and is easy to recognize on the noisy recordings; and (2) the doubly inte- grated area under the central line, which can be expressed by the peak-to-peak height of the first derivative curve, represents 4/16 of the total area (compare with the signal of guaiacol phenoxy-radical (12), which has 21 lines and of which the area under a maximal line represents only 6164 of the total).

The spectra shown in Fig. 5 were obtained by changing the flow rates, and the time courses of two signals, a broad MetHb radical and a multiplet phenoxy-radical, could be measured separately. The repre-

%

After mxmg

6.7 msec

IO msec

FIG. 5. Room temperature spectra of H,O, + p-OH-B, obtained at different flow rates. (From top to bottom: 6.7, 10, 11, 13, and 19 ms after mixing the solutions; MetHb(lOO ~~)-H,Od100 mM) + p-OH- B(10 mM). Conditions:field sweep 100 G/2 min, modulation 100 kHz, 2 G, incident microwave power 10 mW; at 17°C. (The mark indicates the position of the center of ON-(SO,), signal).


sentative time courses are shown in Fig. 6, i.e., (1) the formation rate of MetHb radical increases as increasing H,Oz concentrations by decreases upon addition of p- OH-B; (2) the formation rate of phenoxy- radical increases as increasing the concentrations of HzOz or p-OH-B; (3) the MetHb radical seems to be replaced by the phenoxy-radical, but the decrease in MetHb radical concentration is always greater than the increase in phenoxy-radical concentration at any conditions; and (4) concerning phenoxy-radical formation rate, Michaelis-Menten-type mechanism seems to be operative (Fig. 7). The kinetic con-

(H20zl .25mM [email protected] (H,O,j. IOOmM

I , I .

;1/‘, :‘“, Ii, 0 10 20 0 IO 20 0 10 20

(m set)

FIG. 6. Representative time courses of MetHb radical and phenoxy-radical of p-OH-B. (Top) MetHb radical, (bottom) phenoxy-radical. (0) absence of p-OH-B, (0) presence of p-OH-B. MetHb(lOO PM), H,O, (shown in the Figure), p-OH-B (10 mM). Condi- tions: same as in Fig. 5.

The relative concentrations of phenoxy-radicals and of MetHb radicals are represented by the peak- to-peak heights of the central hyperfine line and of the broad signal, respectively. The heights of MetHb signal are estimated by tracing the base line of the signal of the phenoxy-radical, when two signals over- lap. The relative concentrations are then converted to the concentrations (in PM) on the basis of an addi- tional experiment, which compares the doubly inte- grated area of the spectrum of Fremy’s salt (of known concentration) with those of the MetHb radical and of the phenoxy-radical (the spectra are obtained with the same solutions at the same flow rate as one of the kinetic experiments, under the instrumental conditions of slow field sweep and of large time constant).

stants are summarized in Table II (note that the errors in concentration measurements will be about 20%, due to a poor S/N ratio and to an overlapping of two spectra).

These observations suggest the following reaction scheme which involves an unidentified intermediate complex, X:

MetHb + H,Oz e X- MetHb radical X + p-OH-Bdphenoxy-radical + MetHb (?)

The above scheme is certainly oversimpli- fied, since we are not aware of all elemen- tary processes, for example, the observed decay reactions of MetHb radical and phenoxy-radical are not included.

The decay rate of phenoxy-radical is apparently faster than that of MetHb radical, as can be seen in Fig. 6. In fact, the turn over number for phenoxy-radical formation is smaller than that for MetHb radical formation. The phenoxy-radical de- cays possibly via (1) dimerization of two phenoxy-radicals, (2) reaction with excess H,O, or even (3) reaction with MetHb and/or its radical. Therefore, the kinetic constants for phenoxy-radical formation may well be underestimated.

(c) MetHb + H,O, and benzenoid substrates (Figs. 8 and 9): Extending the

IH*O,I lmMi I~zOdC M) ‘m

FIG. 7. Double reciprocal plot: the formation rates of (a) MetHb radical and (b) phenoxy-radical vs H,O, concentrations. Conditions: same as in Fig. 5.

TABLE II

KINETIC PARAMETERS, MetHb RADICAL AND PHENOXY-RADICAL FORMATION vs H,O,

CONCENTRATIONS

K, V,,,‘,,Jheme

MetHb radical Phenoxy-radical

40 mM 40s’ 200 mM 2os-’


a) /- -.*

Q : i H,C 0 ‘.*a 0%

0.

4 CH.

C% 0.

%C ,--.

g

CH, : ‘.-’

W C”. 0.

’ ’

FIG. 8. Room temperature esr spectra of phenoxy-radicals of (aj 2,6-dimethoxy-, (b) 2,6- dimethyl-, (c) 3,5-dimethyl-, (d) 2,4,6-trimethyl-, and (e) 2,3,5,6-tetramethyl-phenols, produced by MetHb-H,O, system. MetHb(103 FM)-H,O* (25 mM) + phenols(saturated). Conditions:field sweep 100 G/4 min, modulation 100 kHz, 0.5-0.8 G, incident microwave power 10 mW; at 17°C.


FIG. 9. Room temperature esr spectra of naphthoxy-radicals of (a) l-naphthol, (b) 2-naphthol, (c) 2,7-dihydroxy-naphthalene and (d) 2-naphthol-6sulfonate, produced by MetHb-H,O1 and HRP-H,O, systems. (a,b,c) MetHb(213 NM), (d) HRP(0.43 PM)-H,02(100 rn@ + naphthols (saturated). Conditions:field sweep 100 G/2-4 min. modulation 100 kHz, 0.5-0.8 G, incident microwave power 10 mM; at 17°C.

previous results (121, phenols of rather large size were tested. As shown in Figs. 8 and 9, also summarized in Table I, a number of large planary molecules was oxidized to corresponding phenoxy- or naphthoxy-radicals. These radicals are free in solution and not complexed with protein, since (1) hypertine lines are sharp and (2) theoretical calculations agree well with observed spin densities.

p-nitro- and 2,6-dinitro-phenols and benzoic acid gave no signal.

DISCUSSION

Difference in the Reactive Species Involved in HRP-H,O, and

FerroHRP-0, Systems

The most important finding of our rapid freezing experiments is the difference of free radical species, which are produced from the same aromatic molecule during the reactions HRP-H,O, and ferroHRP-OZ. The HRP-H,O, system oxidizes phenols (but not benzoate) and produces phenoxy-

ESR OF HRF’ AND MetHb REACTIONS 477

radicals, as proved by room temperature experiments. While ferroHRP-O2 system yields other type of radicals than phenoxy- radical from both phenols and benzoate.

Many reports have agreed that the HRP compound-III and the oxy-(ferro)peroxidase are the same (3) and this compound is main reactive species existing in the reaction HRP-DHF-0, + benzenoid substrates. HRP-DHF-0, system has been shown to react with phenols and benzoates, finally giving hydroxylated products (1, 2). In addition!, the system is said to possess similar characteristics to Fenton’s reagent, as judged from an absence of NIH shift in the hydroxylation process (32). We have already demonstrated that hydroxy- cyclohexadienyl radicals are produced from benzoates during the reaction of Fen- ton’s reagent at pH 5-7 (12, 19).

The intermediate organic free radicals, detected in the reaction ferroHRP-O2 + benzenoid substrates, may thus be hydroxy-cyclohexadienyl radicals, which would be a plausible structure yielding hydroxylated products. On the basis of the room temperature esr spectra of the radicals (12, 19)1, the following facts must be taken into consideration.

(1) The peak-to-peak width (AH = 14 G) of the #spectrum obtained from the frozen solution of ferroHRP-0, + p-OH-B is much broader than that from phenoxy- radical (AH = 6 G). Such a broad width may reflect an anomalously large hyperfine splitting constant of a proton attached to an sp3-carbon of hydroxy-cyclohexadienyl radicals (of which the splitting constants are some 30 G) (19, 33, 34), while those of the phenoxy-radicals are less than 15 G (12, 24, 25)).

(2) The anisotropy of principal g-values of hydroxy-cyclohexadienyl radical may be greater than that of phenoxy-radicals, because of a considerable asymmetry in the odd electron distribution.

(3) The difference in the average g-values of the phenoxy-radical and of the hydroxy-cyclohexadienyl radical of p- OH-B, is too :small (19) to be differentiated on a frozen spectrum.

(4) Though the saturation behavior against the incident microwave power dif-

fers each other as shown in Fig. 2, the difference in relaxation times does not help the identification of a radical.

As a whole, we suggest the presence of hydroxy-cyclohexadienyl radicals in the reaction ferroHRP-0, + benzenoid sub- stances. However, definite identification of the molecular structure of the radical will require a room temperature experiment.

Similarity of the Reactive.Species Involved in HRP-H202 and MetHb-H,O,

Both systems similarly produce free phenoxy-radicals from phenols, but give no detectable radical from p-nitro- and 2,6- dinitro-phenols and benzoate. The detection of the phenoxy-radicals in the early stage of the reaction may not directly mean that these radicals are the initial products of the reaction, since hydroxy-cyclohexadienyl radicals, probable products for example, are shown to decompose to phenoxy-radicals or even to semiquinone radicals (12, 35, 36). However, hydroxy- cyclohexadienyl radicals from phenols are stable enough to be detected by the present technique at pH 5, even in the presence of excess H202 and iron ions (12). Also, if Fenton-like reaction is operative, there is no reason why the radicals of nitrophenols and benzoate cannot be detected, since the theoretical reactive indices of these molecules (especially free valences) are much the same as p-OH-B, etc. (19). Therefore, we conclude that these phenoxy-radicals are the unique intermediate in these reaction systems.

Since our apparatus is unsuitable for carrying out a stopped flow experiment, we have abandoned kinetical studies on HRP-H,02 system. While Piette et al. have succeeded to separate kinetically the reactions of compound-I and -II, monitoring an esr signal of the chlorpromazine radical (which seems to be rather long- lived and less reactive radical compared with phenoxy-radicals).

In MetHb-H,O, system, we propose the existence of a reactive complex between MetHb and HzOz, denoted as X in the preceding section. X possesses similar re- activity to compound-I and/or -II of HRP and further changes to MetHb radical in


the absence of the oxydogenic substrates or reacts with phenols yielding phenoxy-radicals.

Mechanism of the Phenol Oxidation Yielding Phenoxy-Radical

The liberation of free OH radical in the MetHb-H,O, system is unfavorable as mentioned previously (12), since (1) no trace of hydroxy-cyclohexadienyl radical is observed and (2) benzoate and nitrophenols give no signal.

The substituted phenol- and naphthol- derivatives are oxidized to corresponding phenoxy- and naphthoxy-radicals. Accord- ing to the X-ray crystallography (37), it is evident that these large molecules cannot enter into the heme-pocket of MetHb. Therefore, the phenoxy-radical formation would not take place inside of the heme- pocket but it would at outside of MetHb. The situation may be the same as the case of HRP-H,O,. Dolphin et al. (38) have proposed a structure of r-cation radical of heme for HRP compound-I, on the basis of its optical and magnetic properties. If a similar structure can be supposed for MetHb-H,O, complex, as denoted X, the phenoxy- and naphthoxy-radical formation on the surface of MetHb can be under- stood, since the edges of heme can contact with substrates. In fact, it has been re- ferred that the reaction between metmyo- globin and H,Oz yields a short-lived reactive complex (39). This complex could be compound-I like reactive species.

Finally, concerning the redox-potential of the systems, the present results may throw light on the nature of the reactive species, following George’s finding (40). The potential of phenols decreases as methyl-groups are substituted (41) and increases as carboxyl- or nitro-groups are substituted into the benzene ring (42). In 1930, Fieser (42) has introduced a concept of critical potential, EC, which may be related to the redox-potential of irreversi- bly oxidized molecules. Among various substrates tested, p-nit,ro- and 2,6-dinitro- phenols possess the highest potential, so that the lack of radical formation is under- stood. EC of 1.4 Vmay be an upper limit of

the critical potential to be oxidized by HRP-H,02 and MetHb-H,O, systems.

In short, phenol oxidation catalyzed by MetHb-H,O, and HRP-H,02 systems takes place independently of the molecular size of phenols or of the reactive indices of any particular ring carbon, but dependently on the redox-potential of phenols.

1.

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