Rhus Succedanea Peroxidase and Its Compounds I and II

Takeshi Sakurai and Hideaki Murayama

College of Liberal Arts and Sciences, Kanazawa University, Kanazawa, Ishikawa, Japan

ABSTRACT

In order to explore the process in which lacquer latex is oxidized and polymerized, peroxidase (RSP) was purified from Taiwanese lacquer (Thus succeduneu) latex and characterized. The absorption and magnetic circular dichroism (MCD) spectra of ferric and ferrous peroxidase are quite similar to those of Chinese lacquer (Thus uernicifera) peroxidase (RVP), while spectral features of lactase contained in Taiwanese and Chinese lacquer are considerably different. Exogenous small ligands such as NO, CN-, and CO gave the MCD spectra characteristic to low-spin heme, indicating that these molecules &an occupy an axial position. In addition, the electron spin resonance (ESR) spectra of NO-ferrous enzyme provided strong support for an imidazole group as the proximal (fifth) ligand. The treatment of native enzyme with H,O, gave rise to compound I with a half-life of 10 min. Compound Iwas relatively stable (~r,r = 10 min) but changed to compound II, giving the isosbestic points at 394 and 580 nm. This compound II returned to the resting state with a half-life of 165 min, giving the isosbestic point at 413 nm. The MCD spectra of compounds I and II were similar to those reported for horseradish peroxidase (HRP).

INTRODUCTION

In East Asia lacquer latex has been utilized to form protective thin films for thousands of years. The major components contained in latex are phenol-lipids; urusiol in Japanese and Chinese lacquer, laccol in Vietnamese and Taiwanese lacquer, and thitsiol in Thailander and Burmese lacquer (Melanon-hoea usitata) [l. 21. While it has been revealed that lactase participates in the initial process in oxidizing the diphenol substrates to quinones, it has not been clarified how quinones are polymerized. Recently, we have isolated and spectroscopically characterized RVP [3]. In this communication, we report the spectral properties of RSP, its derivatives with small ligands (NO, CN-, and CO), and the active

Address reprint requests and correspondence to: Dr. Takeshi Sakurai, College of Liberal Arts and Sciences, Kanazawa University, l-l, Marunouchi, Kanazawa, Ishikawa 920, Japan.

Joumal of Inorganic Biochemistry, 48,299-304 (1992) 299

0 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$5.00

300 T Sakurai and Hideaki Murayama

species (compounds I and II). Since content of lactase in Taiwanese lacquer latex is extremely low compared to that in Japanese lacquer latex, contribution of peroxidase together with lactase might not be neglected if an enzyme system which can produce a kind of peroxide is involved. In line with this, we reported that RVP has the activity to oxidize isoeugenol ca. four times faster than Rhus vernicifera lactase [4].

MATERIALS AND METHODS

Fresh Taiwanese lacquer latex was supplied by Takano and Co., Kanazawa, Japan. All chemicals used were of the highest quality commercially available.

RSP was purified according to a similar method to isolate RVP [3]. The SDS gel electrophoresis showed a single band on protein staining, giving a value of Mr = 45000. The molecular weight was slightly smaller than that of RVP (55000) [3] but was slightly greater than that of HRP (40000). Asoret/AZxo value was 2.8, being identical with the purity index of RVP. Heme content was determined by the pyridine hemochrome method [5].

The electronic spectra were measured on a JASCO Ubset50 spectropho- tometer or on an Otsuka Denshi MCPD-1000 spectrophotometer with photodi- ode array detector. In the latter cases, four sets of data acquired for 160 msec were averaged. The MCD spectra were measured on a JASCO J-500C spec- tropolarimeter equipped with a data processor DP-SOON and an electromagnet (1.15 T) at room temperature. The measurements for the CO and NO deriva- tives were anaerobically performed with a Thunberg tube attached with a l-cm path length cell. The ESR spectra were measured on a JEOL RElX X-band spectrometer at 77 K using an ESR tube with a three-way stopcock at its head.

Phosphate buffer (pH 6.0 or 7.2) was used throughout measurements. Water was deionized and distilled.

RESULTS AND DISCUSSION

The absorption and MCD spectra of native ferric and ferrous RSP are displayed in Figure 1. The reduced form of RSP was prepared by addition of a small amount of dithionite. The absorption and MCD features showed a striking resemblance to those of RVP [3], HRP [6-91, and sperm whale myoglobin (Mb) [lo]. It has been proven that the electronic state of ferric-enzyme is an equilib- rium mixture of high-spin heme (major species) and low-spin heme (minor species) at room temperature [3]. The shoulder near 395 nm in the MCD spectrum belongs to the high-spin state and the S-shaped feature in the Soret band is attributed to the low-spin state. On the other hand, the MCD band of ferrous-enzyme comes from the Faraday C term and can be assigned to the high-spin state [ 111.

The treatments of ferric and ferrous RSP with NO and CN- (X 1000) and the treatment of ferrous RSP with CO also gave the analogous spectra shown by RVP [3l (spectra not shown). MCD spectra clearly indicated that the derivatives treated with these small molecules have a heme in the low-spin form. Neverthe- less, this does not necessarily mean that an exchangeable water molecule is coordinated at an axial position of HRP as in cytochrome c peroxidase and Mb [12, 131. The second derivative of the ESR spectrum of NO-treated RSP showed

0

60

coMPouNDsI ANDIIOF ~AC~UERPEROXIDASE

1 n a

300 400 500 600 700

Wavelength (nm)

10

-20

301

300 400 500 600 700

Wavelength (nm)

FIGURE 1. Absorption (a) and MCD (b) spectra of ferric (solid line) and ferrous (broken line) RSP (5.0 PM) in 0.05 M phosphate buffer (pH 6.0).

a triplet of triplets superhyperfine structure (g, = 2.073, g,. = 2.040, g, = 2.004, g,=2.001, A,= 2.04 mT, A,, = 0.67 mT), indicating that a histidine imidazole group is the proximal (fifth) ligand [3].

Compounds I and II are active intermediates which appear in the course of the enzyme reaction.

Peroxidase + H,O, -+ Compound I + H,O,

Compound I + AH + Compound II + A= + H+,

Compound II + AH + H+ + Peroxidase + A- + H,O,

302 T. Sakurai and Hideaki Murayama

in which AH and A= are substrate and free radical, respectively. Compound I, which is now known as (R= + )Fe(IV) = 0 (R. + is a porphyrin rr cation radical) [14, 151 was formed soon after addition of 2H,O, to the resting peroxidase. lH,O, is required to form compound I, but 2H,O, was needed to ensure complete formation of it. In the case of RVP, this intermediate was too unstable to be measured its absorption spectrum, however, compound I of RSP has a considerably long life time (~i,~ = 10 min) at room temperature. The absorption spectrum (Fig. 2) showed maxima at 400 (8 = 50000), 563 (E = 6500), 600 (6 = 6400), and 660 (6 = 5400) nm. The absorption feature seems to be similar to that of milk peroxidase, which showed the absorption maxima at 410, 562, 600, and 662 nm [16], rather than the well characterized one of HRP peroxidase, which showed the absorption maxima at 400, 577, 622 and 651 nm [8]. Com- pound I changed to compound II, giving the isosbestic points at 394 and 580 nm (Fig. 2). Th e r su mg compound II in which the porphyrin accepted an electron e It’ (RFe(IV) = 0) 1171, gave the absorption maxima at 418 (E = 83000), 532 (E = 95001, and 554 (E = 9500) nm. This absorption feature was very similar to those of HRP (420, 527, and 554 nm) [8, 18, 191 and RVP (418, 533, and 558 nm) [3] but was slightly different from that of milk peroxidase (433, 537, and 568 nm) [16]. Finally, compound II returned to the original resting form with a half-life of 165 min, showing a pseudo-isosbestic point at 413 nm.

MCD spectra of compound I was obtained by scanning from 700 to 350 nm at as fast as 100 nm/sec rate (Fig. 3). The spectral feature seems to be consider-

100

4

‘E AU

‘J w

50

0 350

ferric peroxidase

400 450 500 600 700

Wavelenqth(nm) wavelength lnm)

500 600 700

Wavelength lnm)

10

I

B 3

‘2 5 "

FIGURE 2. Absorption spectra of RSP (5.0 PM) in the presence of H,O, (10 PM) in 0.1 M phosphate buffer (pH 6.0). Ferric peroxidase, compound I (5 set> and compound II (54 min) are indicated by solid lines and intermediates (3, 7, 11, 19, 91, 150, 250, and 338 min) by broken lines.

COMPOUNDS I AND II OF LACQUER PEROXIDASE 303

-20 /- -l-l0

t I 1 I 1 400 500 600 700

Wavelength (nm)

FIGURE 3. MCD spectra of compound I (broken line) and compound II (solid line) of RSP (5.0 PM) measured at magnetic field, 1.15 T.

ably similar to that reported for the frozen compound I of HRP measured at 77 K 181, although the MCD band of compound II superimposed on the Soret band (17%), gave a shoulder at 423 nm. According to the temperature dependence study, the MCD spectrum of HRP compound I has been attributed to the S = 3/2 Kramer’s doublet ground state of the coupled Fe(IV) and r-cation radical species (15 + 5 cm-‘) [8, 18, 193. We speculate that compound I of RSP is also in the S = 3/2 ground state with a similar magnitude of zero-field splitting. The MCD spectrum of compound II shows the S-shaped bands in Soret and Q regions (Fig. 31, which are assigned as a Faraday A term from the MCD spectra of HRP compound II [8, 18, 193.

In the future, we will compare the reactions of peroxidase and lactase for a variety of substrates.

This study was part& supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Mnistry of Education, Science, and Culture of Japan (No. 03241210).

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304 T. Sakurai and Hideaki Murayama

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Received March 1 I, 1992; accepted May 28, 1992