The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: An electron spin resonance investigation

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 273, No. 1, August 15, pp. 15%164,1989

The One-Electron Oxidation of Porphyrins to Porphyrin Pi-cation Radicals by Peroxidases: An Electron Spin Resonance Investigation KIM M. MOREHOUSE,’ HERBERT J. SIPE, JR.: AND RONALD P. MASON3 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, PO Box 12233,Research Triangle Park, North Carolina 27709 Received January 3,1989, and in revised form April 17,1989

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, AH = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III > coproporphyrin I > deuteroporphyrin IX > mesoporphyrin IX > Photofrin II > protoporphyrin IX > uroporphyrin I > uroporphyrin III > hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration. o 1989 Academic POW, I~~.

Porphyrins are aromatic tetrapyrrolit macrocycles. Iron protoporphyrin IX (heme) is the central component of hemoglobin, myoglobin, catalase, peroxidases, and many of the cytochromes. The participation of heme proteins in the oxygen transport, peroxide reduction and disproportionation, the mitochondrial electron transport chain, and drug metabolism (cytochrome P450) stresses the biological importance and diverse roles of the iron porphyrins (1). The formation of a porphyrin 1Current address: Division of Food Chemistry and Technology, Food & Drug Administration, 200 C Street, S.W., Washington, DC. 20204. aPermanent address: Department of Chemistry, Hampden-Sydney College, Hampden-Sydney, VA 23943. 3To whom correspondence should he addressed. 0003-9861/89 $3.00 Copyright 0 1989 hy Academic Press, Inc. All rights of reproduction in any form reserved.

pi-cation radical of the prosthetic group of horseradish peroxidase occurs when this enzyme reacts with hydrogen peroxide to form the classic compound I ((2) and references cited within). In this oxidation of the heme, the ferric ion is oxidized to the ferry1 form and the porphyrin to its pi-cation radical. Two types of electronic structures for the porphyrin radical, alu and a~~,have been proposed for catalase and horseradish peroxidase, respectively, and the relation between the structure and catalytic function has been discussed (3,4). Considerable emphasis has been placed on the role of the central metal ion. However, the universality of metalloporphyrins in nature and the many varied functions they perform suggest that besides binding the metal ion at an appropriate site in the protein or modifying the redox 158

OXIDATION

159

OF PORPHYRINS BY PEROXIDASES TABLE I

ESR PARAMETERS FOR THE PORPHYRIN PI-CATION RADICALS GENERATED BY HORSERADISH PEROXIDASE Porphyrin

Q (*o.oOOl)

Coproporphyrin III Coproporphyrin I Deuteroporphyrin IX Mesoporphyrin IX Photofrin II Uroporphyrin I Uroporphyrin III Protoporphyrin IX Hematoporphyrin IX

2.0024 2.0024 2.0024 2.0024 2.0023 2.0023 2.0023 2.0024 -

potential of the metal, the porphyrin ring itself exhibits properties necessary for proper biological function. The enzymatic redox properties of metal-free porphyrins may be of significance in porphyria and in the use of porphyrins as photosensitizers in tumor phototherapy (5-11). The porphyrias are a family of disorders associated with alterations of the activity of one or more of the enzymes involved in heme biosynthesis. Depending upon which enzyme is malfunctioning, several different porphyrins can build up in different portions of the body causing various pathological consequences (12-16). With tumor phototherapy (photodynamic therapy), large concentrations of porphyrin (or porphyrin-like compounds) are injected at the site of the tumor, causing a localized high concentration of porphyrin which may slowly disperse throughout the patient’s body. The biological consequences of these high porphyrin concentrations may be similar to those of porphyria patients. In previous papers (17,18) we have presented data on the enzymatic one-electron reduction of some metal-free porphyrins to their respective anion free radicals in an effort to understand the possible biotransformations of porphyrins. We present here the enzymatic oxidation of several naturally occurring, as well as synthetic, porphyrins as catalyzed by several peroxidases. This work demonstrates that porphyrins can be substrates for peroxidases, and that the oxidation is,

AI&

(G, +0.2) 2.8 2.6 3.1 3.3 3.7 2.3 2.4 5.3 -

Relative peak height 1000 789 3% 232 103 18 10 8 No signal

for the most part, facile. Furthermore, we identify for the first time that the enzymatic one-electron oxidation product of metal-free porphyrins is a porphyrin pication radical. Enzymatic oxidation of porphyrin also results in changes in the visible absorption spectra that are caused by the formation of dihydroxyporphyrin, a twoelectron oxidation product. MATERIALS

AND METHODS

Horseradish peroxidase (Type VI), lactoperoxidase, hydrogen peroxide (30%), and DTPA4 were obtained from Sigma Chemical Co. Myeloperoxidase was obtained from Calbiochem (Behring Diagnostics). Photofrin II was from Photomedica, Inc. (obtained as a 2.5 mg/ml saline solution and diluted with buffer to the final concentration). All other porphyrins were obtained in their highest purity from Porphyrin Products and used without purification. Some of the porphyrins were first solubilized in a 0.1 M KOH solution and then diluted to the final concentration with buffer. Where possible, the porphyrins were solubilized directly in the buffer at the desired concentration by stirring. Where appropriate, the porphyrins were protected from light to minimize any effects due to photoreduction (6, 17). All other chemicals were of the highest purity available and were used as obtained. ESR observations were made at room temperature with an IBM ER200D spectrometer operating at 9.7 GHz with RIO-kHz modulation frequency and equipped with an ER-4103 TM cavity. The g-factors

4Abbreviation used: DTPA, diethylenetriaminepentaacetic acid.

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MOREHOUSE, SIPE, AND MASON

were measured relative to Fremy’s salt (Q = 2.00550 f 0.00005) as previously described (19). Solutions were transferred to the quartz flat cell by means of a rapid sampling device (Gilford Instruments) (20). This means that the tedious tuning of the flat cell had to be done only once, and ensured that the cell remained in the same position for the g-factor determinations. Optical absorption spectra of the porphyrins and their oxidized products were recorded with a Hewlett-Packard 8451A photodiode array spectrophotometer. Spectra were recorded with a l-cm path length cell and a l-s accumulation time.

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,

I

URok’ot?PHYRlN HRP H202

JGAUSS

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-FEROXIDASE

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I

RESULTS

The enzymatic one-electron oxidation of several synthetic and naturally occurring porphyrins was investigated (Table I). All of the porphyrin pi-cation radicals have ESR spectra characterized by a broad (-3 G) singlet with ag-factor of 2.0024. Similar ESR spectra of porphyrin cation radicals (g = 2.0025 + 0.0001) can also be detected by the electrochemical oxidation of porphyrins in organic solvents (4,21). Figure 1A displays the ESR spectrum obtained upon the one-electron oxidation of uroporphyrin I. If porphyrin (Fig. lB), horseradish peroxidase (Fig. lC), or hydrogen peroxide (Fig. 1D) is omitted, no signal is detected. Uroporphyrin I is currently under investigation for its possible use in photodynamic therapy (22-25). We have previously demonstrated that uroporphyrin I can be enzymatically reduced to a porphyrin anion radical (1’7,lS). A synthetic porphyrin which has received much attention in recent years as a tumor photosensitizing agent is Photofrin II. Figure 2 displays the ESR spectrum observed upon the oxidation of Photofrin II by horseradish peroxidase. Photofrin II pication radical formation is also dependent on the presence of horseradish peroxidase and hydrogen peroxide (data not shown). The enzymatic oxidation of several porphyrins by horseradish peroxidase was investigated (Table I), and coproporphyrin III was found to give the strongest ESR signal (to have the highest steady-state radical concentration). Again, the porphyrin pi-cation radical signal is completely

FIG. 1. Electron spin resonance spectrum of the porphyrin pi-cation radical generated by the oneelectron oxidation of uroporphyrin I by the horseradish peroxidase system. (A) An aerobic incubation containing 0.5 mM uroporphyrin I, 0.1 mg/ml horseradish peroxidase, 50 pM hydrogen peroxide in a 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM DTPA. (B) Same as A, but in the absence of uroporphyrin I. (C) Same as A, but in the absence of horseradish peroxidase. (D) Same as A, but in the absence of hydrogen peroxide. Spectrometer settings: microwave power, 21 mW; modulation amplitude, 2 G; sweep rate, 0.04 G/s; time constant, 2 s; gain, 1.6 x 106.

dependent on porphyrin, horseradish peroxidase, and hydrogen peroxide (data not shown). A higher steady-state concentration of porphyrin pi-cation radical is obtained for Photofrin II than for uroporphyrin I (Table I). This is in contrast with the enzymatic reduction of these two porphyrins (18), where uroporphyrin I gave the more intense ESR signal. Although horseradish peroxidase-catalyzed reactions serve as a good model for peroxidases, this enzyme is of plant origin. Therefore, two mammalian peroxi-

OXIDATION

161

OF PORPHYRINS BY PEROXIDASES

PHOTOFRIN II HRP H202

FIG. 2. ESR spectrum of the porphyrin pi-cation radical generated by the one-electron oxidation of Photofrin II by an aerobic incubation containing 0.1 mg/ml Photofrin II, 0.1 mg/ml horseradish peroxidase, 50 CMhydrogen peroxide in a 100 mM potassium phosphate buffer (pH 7.4) with 0.1 rnM DTPA. Spectrometer settings: microwave power, 21 mW, modulation amplitude, 1 G; sweep rate, 0.05 G/s; time constant, 2 s; gain, 5 X 105.

dases, lactoperoxidase and myeloperoxidase, were also investigated. Consequently, several of the porphyrins were also oxidized to their pi-cation radical with these enzymes. As expected, identical spectra were obtained. Figure 3 displays the results obtained by the oxidation of coproporphyrin III as catalyzed by myeloperoxidase, with similar results obtained for lactoperoxidase (data not shown). The ESR signal is again completely dependent on the porphyrin, the mammalian peroxidase, and hydrogen peroxide (data not shown). To further characterize the enzymatic process, the concentration of porphyrin CC!PROPORPHYRlN Ill MYELOPEROXIDASE

FIG. 3. ESR spectrum of the porphyrin pi-cation radical generated by the one-electron oxidation of coproporphyrin III by an aerobic incubation containing 0.1 mM coproporphyrin III, 0.5 U/ml myeloperoxidase, 50 pM hydrogen peroxide in a 100 mbi potassium phosphate buffer (pH 7.4) with 0.1 mM DTPA. Spectrometer settings: microwave power, 21 mW, modulation amplitude, 2 G; sweep rate, 0.05 G/s; time constant, 2 s; gain 1 X 10’.

,/[HAP

(mg/ml)]

FIG. 4. Horseradish peroxidase dependence of the uroporphyrin I pi-cation radical concentration. The steady-state radical concentration was proportional to the square root of the horseradish peroxidase protein concentration, thus indicating that the radical decay is kinetically second order and nonenzymatic. Other experimental conditions and spectrometer settings were as in Fig. 1.

was varied while the concentration of horseradish peroxidase and hydrogen peroxide were held constant. As the concentration of porphyrin increases, the steadystate ESR signal intensity also increases. The ESR signal reaches a steady-state level in several minutes then decays, presumably due to the depletion of hydrogen peroxide. We also investigated the effect of varying the concentration of the horseradish peroxidase on the steady-state ESR signal intensity (Fig. 4). The steady-state concentration of the porphyrin pi-cation radical was not linear with respect to the horseradish peroxidase protein concentration but was proportional to the square root of the protein concentration, indicating that the radical decay is kinetically second order. This implies nonenzymatic disproportionation or dimerization of the radical (26). Since porphyrins have intense and characteristic absorption spectra, we also measured changes in the absorption spectra of the porphyrins during enzymatic oxidation. Although we were unable to detect the weak optical absorbance expected for the pi-cation radical, a broad peak around 700 nm (2’7-29), we were able to see a change in the absorption spectra associ-

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MOREHOUSE, SIPE, AND MASON

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WAVELENGTH hm)

PHOTOFRIN P

WIMLENGTH

hm)

FIG. 5. Optical absorption spectra for the oxidation of porphyrins by horseradish peroxidase. (A) Reaction mixture contained (-) 10 g&i coproporphyrin III and 0.1 mg/ml horseradish peroxidase; (---) spectra taken 38 min after the addition of 100PM hydrogen peroxide; (. . .) 0.1 mg/ml horseradish peroxidase and 100 CM hydrogen peroxide. (B) Reaction mixture contained (-) 10 PM Photofrin II and 0.1 mg/ml horseradish peroxidase; (---) spectra taken 115 min after the addition of 100 PM hydrogen peroxide; (. . . ) 0.1 mg/ml horseradish peroxidase and 100 pM hydrogen peroxide. All of the samples were prepared in a 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM DTPA. The spectra were recorded with a l-cm path length cell and a l-s acquisition time.

ated with another oxidation product. Figure 5A displays the results obtained for coproporphyrin III, and Fig. 5B displays the results for Photofrin II. The solid line is due to the parent porphyrin, and the change in the spectrum (dashed line) is due to the formation of product(s). The dotted line is due to the protoporphyrin IX of the peroxidase. For coproporphyrin III, similar results were obtained when lactoperoxidase was used in place of horseradish peroxidase. The absorption at 630 nm has

been detected previously as a decay product from other porphyrin pi-cation radicals and has been assigned as the dihydroxyporphyrin, a two-electron oxidation product (30,31). This chromophore can be bleached upon further oxidation. Thus the dihydroxyporphyrin is just one intermediate involved in the oxidation process. Although further changes in the absorption spectra could be detected, no other products were identified. The same spectral changes were found upon chemical oxida-

OXIDATION

OF PORPHYRINS

tion (with Brz), thus demonstrating that the changes in the absorption spectra are indeed due to the oxidation of the porphyrin. DISCUSSION

Although metalloporphyrin pi-cation radicals are known to occur as intermediates in several enzymatic processes, the formation of these radicals by peroxidases acting on metal-free porphyrins as substrates has not been reported. From Table I, it can be seen that the relative steadystate concentrations of the porphyrins varied considerably from coproporphyrin III to hematoporphyrin IX, which is undetectable. The differences in the steadystate concentrations of the porphyrins can be due to several chemical properties: (i) differences in their oxidation potentials, (ii) rates of decay of the pi-cation radicals, (iii) aggregation of the porphyrins, and (iv) steric and/or ionic hindrance. It has been shown by pulse radiolysis (30,31) that the porphyrin pi-cation radical decays via a disproportionation process, yielding the corresponding porphyrin di-cation: 2 HsP =

2 H,P’+

=

H,P

+ H*P’+

The porphyrin di-cations are strong electrophiles which react with water to form isoporphyrins (32) and mesodihydroxyporphyrins (33): These products are unstable and undergo further reactions yielding ring opening, resulting in destruction of the porphyrin chromophore and bleaching of the absorption spectra. During the course of enzymatic oxidation, we were able to obtain an absorption spectrum differing from that of the parent porphyrin. From the absorption spectrum, we have tentatively assigned this product to a 5,6-dihydroxyporphyrin, on the basis of its absorption at 630 nm (31). The experimental results presented here demonstrate that the enzymatic one-electron oxidation of porphyrins yields por-

BY PEROXIDASES

163

phyrin pi-cation radicals. Thus, we demonstrate that porphyrins are substrates for peroxidases. The formation of the porphyrin free radical is dependent on peroxidase, hydrogen peroxide, and substrate. Several peroxidases are capable of facilitating this oxidation process; horseradish peroxidase, lactoperoxidase, and myeloperoxidase were used in this study. These results, along with our previously reported data on the enzymatic one-electron reduction of porphyrins to their corresponding anion radicals (17,18), demonstrate that porphyrins are capable of undergoing metabolic reactions other than those usually associated with heme metabolism (32).

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