Complexes of iron and cobalt tetrasulphonated phthalocyanines with horseradish peroxidase protein

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Pdyhimm Vol. 9, No. 8, pp. 1021~1026.1990 Printedin Great Britain

COMPLEXES OF IRON AND COBALT TETRASULPHONATED PHTHALOCYANINFS WITH HORSERADISH PEROX~ASE PROTE~ LUCJA OSTROPOLSKA Institute of Chemistry, University of Wroclaw, Poland

Abstra&--The interaction between the iron and cobalt tetrasulphonat~ p~thalocyanines and the horseradish peroxidase protein leads to the formation of artificial peroxidase containing phthalocyanine derivative in place of protohaem. Such a modification has also been carried out by replacement of the haem in peroxidase by metal tetrasulphonated phthalocyanine. The complexes are not formed with apo-peroxidase previously modified by carbox~ethylation at histidines. This suggests that the binding site of iron and cobalt phthalocya~nes is a histidine residue, as is the case of the haem in the native peroxidase. The visible abso~tion spectra show the main peak of the iron compound at 658 nm and that of the cobalt compound at 680 nm. The position of the main band and the properties of the cobalt compound suggest three oxidation states of the cobalt ion. It is shown by circular dichroism (CD) experiments that incorporation of Fe”‘L or Co”‘L into the peroxidase protein leaves the helical content of the protein virtually unchanged in spite of their larger sizes. It is, however, markedly lower than that of the native enzyme, which points to the lower stability of the model complexes. Molecular weight results indicate that the synthesized compounds are monomer. Both pbthalocyanine-mod~ed peroxidases are reduced by dithionite and ferr~ytochrome c to give iron(I1) and cobalt(I1) complexes which are able to undergo reversible oxygen binding. Artificial peroxidases exhibit minimal enzymic activity and for this reason they cannot be regarded as physiological models of the native peroxidase.

The important role of metalloenzymes in the biological processes has led to the synthesis and study of their model analogues. One of the most important classes of metalloenzymes constitute the haem proteins. They are formed by conjugation of proteins with iron porphyrin which plays the role of the prosthetic group. ’ The interaction between the

Abb~viations : L : tetrasulphonated phthalocyanine ligand, C&3, 2Ns (S%Na),. Fe’nL : iron(II1) tetrasulphonated phthalocyanine. HRP : horseradish peroxidase. apo-peroxidase : peroxidase protein, without its prosthetic haem group. Fe”‘L-apo-peroxidase : iron(II1) te~sulphona~ phthaIocyanine complex with peroxidase protein. Co’iL : cobalt(I1) tetrasulpho~t~ phthalocyanine. CoinL-apo-peroxidase : cohalt(II1) tetrasulphonated phthalocyanine complex with peroxidase protein.

haem group and the surrounding protein is critical for the functional design of haem proteins.2*3 The interaction has been investigated by comparing the reactivity of the physical properties of model systems with those of the proteins.4*5 The effect of metal substitution on peroxidase co~o~ational stability and the effect of the protein on the haem centre have been reported for the following metallosubstituted peroxidases : CoHRP, Cu-HRP, Mn-HRP and V-HRP.“9 Also, the effect of chemical modification of the porphyrin side chain and the role of peripheral substituents of the porphyrin in metalloporphyrin-apo-peroxidase complexes have been studied.‘&r3 According to these results, the major role in the restoration of the helical content of a protein is played by the porphyrin ring itself, not by the metal component. For functionally active peroxidase a hydrophobic en~ro~ent of the prosthetic groups is required. The compounds which closely resemble metal-

1021

t.

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OSTROPOLSKA

loporphyrins are metal tetrasulphonated phthalocyanines. Recently it has been shown that iron and cobalt tetrasulphonated phthalocyanines are able to displace haem in haemoglobin and myoglobin to give complexes with properties resembling those of haemoglobin and myoglobin.‘4*‘5 Iron and cobalt phthalocyanine-substituted cytochrome c has also been synthesized. The conformation of the protein in those compounds is significantly altered compared to that in the native cytochrome c. These phthalocyanine-substitute compounds are lowspin species with the metal ions in the trivalent state. ’ ~5,’7 In this article we report the results concerning the preparation, structure and properties of artificial peroxidase obtained by incorporation of iron and cobalt tetrasulphonated phthalocyanines into the haem binding site of the peroxidase protein.

materials

and methods

Horseradish peroxidase, type I, was purchased from Sigma Chemical Co. and the isoenzyme C was separated and purified by the method of Shannon et at. 18 The purity index (&,3Rm: A 278m) of this enzyme was 3.4. The apo-peroxidase was prepared from horseradish peroxidase by a modification of Teale’s acid butanone technique reported by Yonetani. I9 The concentration of the solutions was assayed spectrophotometrically using absorption coefficients, based on separate dry weight deter~natio~s : 9.2 x lo4 M- ’ cm- ’ at 403 nm for the native protein and 1.3 x lo4 M- ’ cm-’ at 278 nm for the apo-protein. The preparation of the carboxymethylhistidine derivatives of apo-peroxidase was performed by iodoacetylation at pH 5.5 according to the method described previously.2o The preparation and purification of iron and cobalt tetras~phonated phthalo~yanines have been described by Fallab and co-workers.2’ The stock solution was obtained by weighing an appropriate amount of the solid and dissolving the latter in 100 cm3 of an appropriate buffer.

Co”L with the apo-peroxidase were not adsorbed on DEAE-cellulose, whereas free iron and cobalt tetrasulphonated phthalocyanines were firmly bound to this adsorbent. The complex fraction of the chromatographic separation was concentrated by lyophili~tion and stored at 0°C. The concentrations of the phthalocyanine complexes in the solutions were determined from the molar absorption coefficients : FeiuL-apo-peroxidase &658 = 2.4 x lo4 M-’ cm-’ and Co”‘L-apo-peroxidase &680 = 3.8 x IO’ M-’ cm-‘. Solid samples were obtained by lyophilization of the water solutions of the complexes. The reduced forms of the complexes were prepared by addition of a few milligrams of sodium dithionite to their buffered solutions and removal of the reductant excess on a Sephadex G-50 column. All the chemicals used in the studies were of analytical purity. Absorption spectra and difference spectra were recorded on a Cary 15 spectrophotometer with a cell compartment thermostatically controlled at 4°C or on a Specord spectrophotometer. Molecular weight measurements of the model complexes were carried out by gel-filtration on a Sephadex G-75 column according to the method of Andrews.22 The following proteins were used as reference substances: cytochrome c (mol. wt 12,400), myoglobin (mol. wt 17,800), chymotrypsinogen (mol. wt 25,000), ovalbumin (mol. wt 45,000), serum albumin (mol. wt 67,000) and yglobulines (mol. wt 160,000). Circular dichroism spectra were recorded on a model ORD/W-5 Japan Spectropolarimeter with CD attachment. The solutions of the complexes were prepared by dissolving the appropriate amount of lyophilized preparation in phosphate buffer, pH = 7.4. Enzyme assay

Peroxidase activity was measured spectrophotometrically by following the change in absorbance at 460 nm due to o-dianisidine oxidation in the presence of H202 and the complexes.23 RESULTS

Synthesis of the metal tetrasulphonated ~ya~i~~-~bst~t~tedperoxidas~

phthalo-

The apo-peroxidase in 0.01 M Tris, pH = 8.4, was adjusted to 0.2 mM, mixed with a two-fold excess of iron or cobalt tetrasulphonated phthalocyanine, and allowed to stand at 4°C for 2 days. The mixture was passed through a DEAE-cellulose column equilibrated with 10 m&I potassium phosphate buffer, pH = 7.4. The complexes of Fe**‘L and

The reaction of Fe”‘L and Co’iL with peroxidase indicates that haem is partially displaced by phthalocyanine compounds. This is shown by the difference spectra presented in Fig. 1. In both cases the Soret band at 403 nm, which is characteristic of peroxidase, disappears. The formation of phthalocyanine complexes is monitored by the appearance of a new characteristic band in the region of phthalocyanine absorption at 658 and 680 nm, respec-

Iron and cobalt tetrasulphonated

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Fig. 1. Difference spectra of the mixture of HRP with (a) Fe’uL and (b) Co”L ). Difference spectra of (a) Fe”‘L+apo-peroxidase and (b) solution mixed (oxidase mixtures against the same solution unmixed (---). Spectra were recorded reaction run in 5 mM phosphate buffer, pH = 7.5. Concentrations: oxidase = 1 x lo-’ M, Fe”‘L = Co”L = 2 x lo-’ M.

for iron and cobalt protein complexes. At the same time the characteristic absorption of Fe”‘L at 635 nm and Co”L at 625 nm decreases. The reaction of haem displacement in peroxidase by phthalocyanine derivatives was investigated in the pH range 4.5-9.5. The optimal pH for this reaction appeared to be 5.5. This fact suggested that displacement of haem by phthalocyanine was dependent on dissociation of the peroxidase into haem and apo-protein. 24It is known from previous investigations 25 that tetrasulphonated phthalocyanine exists in aqueous solution in two forms: dimeric and monomeric. The stability of the dimers was found to decrease in the following way: Fe”‘L > Co”L. This could explain the higher displacement reaction rate in the case of Cor’L than that of Fe”‘L and confirms previous suggestions that the monomer is a species which displaces haem from haemoproteins. 26 tively,

Combination of Fe”‘L and Co”L with peroxiakse protein The reaction between metal tetrasulphonated phthalocyanine and apo-peroxidase was demon-

against the same Co”L+apo-perafter 24 h of the HRF’ = apo-per-

strated by a difference absorption spectroscopy experiment (Fig. 1). The difference spectra of both systems in the visible region correspond closely to those of the systems with the native peroxidase. It is evident from these spectra that in the reaction of peroxidase apo-protein with phthalocyanine compounds new complexes are formed which are identical to those arising in the haem displacement process. In contrast to free Cor’L, its complex with apoperoxidase has very little ability to undergo reversible oxygen binding, which suggests that insertion of Co”L into the apo-protein under aerobic conditions leads to oxidation of the cobalt ion to its trivalent state. Spectroscopic titration of an apo-peroxidase with Fe”‘L and Co”L points to a 1: 1 molar ratio of the reagents. The new apo-peroxidase complexes were isolated as green-blue solids by separation of the reaction mixture on DEAE-cellulose and by lyophilization. The solids were stable for 4 weeks. The incubation of iron and cobalt tetrasulphonated phthalocyanines with a carboxymethylhistidine derivative of the apo-peroxidase did not lead to the formation of the complexes.

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t. OSTROPOLSKA

This fact suggests that the metal phthalocyanine binding site in apo-peroxidase is the histidine group. Properties of the Fe”‘L-apo-peroxidase apo-peroxidase complexes

and Co”‘L-

The reduction of the Fe”‘L-ape-peroxidase with dithionite involves direct attack of the reductant on the iron(II1) atom. The spectrum of the reduced form of this complex exhibits a characteristic band at 690 nm. No degradation of the phthalocyanine ring is observed in this spectrum in contrast to that of free Fe”L where disappearance of the phthalocyanine characteristic bands occurs. I4 The results presented above suggest that the Fe”‘L-apo-peroxidase compound is located deeply inside the protein. A solution of Fe”L-ape-peroxidase exhibits reversible combination with oxygen. Absorption spectra of this compound in argon and oxygen are shown in Fig. 2. The reduction of Co”‘L-ape-peroxidase with dithionite in argon results in the disappearance of the band at 680 nm and increased absorptivity at 635 nm. Bubbling oxygen through the solution gives rise to a new band at 690 nm. At the same time the band at 635 nm disappeared. In an argon atmosphere the reaction reverses. These results indicate that the reduced form of the Co”L-ape-peroxidase

1

complex is able to undergo reversible oxygen binding. The band at 690 mn corresponds to the oxygenated form of the complexes and that at 635 nm to its deoxygenated form. The oxygen displacement from the oxygenated form of the iron complex is more difhcult than in the case of the cobalt complex, which suggests a stronger metal-oxygen bond in the iron complex. The difference spectrum presented in Fig. 2 shows that Fe”‘L-ape-peroxidase is able to oxidize ferrocytochrome c under anaerobic conditions. The increase in absorptivity at 406 nm and its decrease at 417 mn points to the transformation of ferrocytochrome c to ferricytochrome c. The simultaneous decrease of absorptivity at 658 run and its increase at 690 nm indicates that Fe”‘L-apo-peroxidase is reduced to Fe”L-ape-peroxidase. Both model complexes reacted slowly with hydrogen peroxide to form “peroxide compounds” with characteristic absorption bands at 662 and 682 nm for Fe”‘L-ape-peroxidase and Co”‘L-apo-peroxidase, respectively (Fig. 3). When catalase was added to the solution, H202 was decomposed and the original spectrum of Fe”‘L-ape-peroxidase and Co”‘Lapo-peroxidase was regenerated. This suggests that H202 coordinates to the metal atoms. However, the intensities of these spectra diminish with time, which is indicative of slow auto-oxidation of the phthalocyanine ligand. The purified Fe”‘L-ape-peroxidase and Co”‘L-ape-peroxidase gave activities of 500 and

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Fig. 2. The optical absorption spectra of (a) Fe”‘L-ape-peroxidas and (b) Co”‘L-ape-peroxidase in sodium phosphate buffer, pH = 7.5 with dithionite. M”‘L-ape-peroxidase (--), M”‘L-apoperoxidase+Na,S,O, in oxygen (---), M”‘L-ape-peroxidase+Na,S,O, in argon (-.-.--). Concentration: Fe”‘L-ape-peroxidase = Co”‘L-ape-peroxidase = 2 x IO-’ M. Difference spectra of the mixture Fe”‘L-ape-peroxidas with ferrocytcchrome c (a) against unmixed solution in anaerobic conditions ( . . -). Concentration of Fe”‘L-ape-peroxidas = ferrocytochrome c = 5 x 10m6M.

Iron and cobalt tetrasulphonated phthalocyanines (a)

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Fig. 3. Absorption spectra of (a) Fe”‘L-ape-peroxidase and (b) Co”‘L-apo-peroxidase in phosphate buffer, pH = 7.5 with hydrogen peroxide. M’nL-apo-peroxidase ), M”‘L-apo-per(oxidase+H,O, after 10 min of the reaction run (---), after 2 h of the reaction run (-.--.-). Concentrations: Fe”‘L-ape-peroxidase = Co”‘L-ape-peroxidase = 5 x 10m6 M, H202 = 5 x 1O-6 M.

350 mol min- ’ mg- ‘, respectively, in the standard oanisidine assay. The results of the molecular weight estimation using Sephadex G- 100 gel-filtration techniques demonstrated that the molecular weight of the iron complex is aproximately 42,500 and that of the cobalt one is 42,000. These values are comparable with that of peroxidase, which indicates that the model complexes are monomers. To obtain information about whether the metal tetrasulphonated phthalocyanine incorporation in the peroxidase protein brings about perturbation of its helical structure, the UV CD spectra of the appropriate mixtures were taken. The results of these experiments, presented in Table 1, indicate that the helical content of the peroxidase apo-protein is virtually unchanged due to metal phthalocyanine coordination. CD spectra of the metal phthalocyanine-modified peroxidases exhibit molar ellipticities close to that of the peroxidase apo-protein. However, they are markedly lower than that of the native peroxidase.

Table 1. Comparison The

medium

of CD results of the complexes. contained 5 mM phosphate buffer, pH = 7.5

Compound Fe”‘L-ape-peroxidase Co”‘L-ape-peroxidase Apo-peroxidase Peroxidase

0 x IO6 ’ cm2 dmol-’ 221 nm 208 nm -3.20 - 3.25 - 2.90 -3.65

-3.75 -3.85 -3.45 -4.15

CONCLUSIONS Interaction between the iron and cobalt tetrasulphonated phthalocyanines and peroxidase protein results in the formation of the complexes Fe”‘L-apo-peroxidase and Co”‘L-apo-peroxidase with characteristic absorptivities at 658 and 680 nm, for iron and cobalt complexes, respectively. The same complexes were obtained by haem displacement in the native peroxidase with Fe”‘L and Co”L. This suggests the same binding site on the protein for haem and phthalocyanine derivatives. The stoichiometry of this process was found to be 1: 1 in the systems with both phthalocyanine complexes but the reaction rate was higher when Co”L was used. This suggests a different mechanism for the process with the cobalt complex. The position of the main absorption band of the cobalt phthalocyanine-modified peroxidase, as well as its lack of ability to undergo reversible oxygen binding, which is characteristic for free Co’IL, points to three oxidation state of the cobalt ion in this compound. According to the suggestions of Hoffman and Petering2’ reconstitution of the cobalt-substituted haemoglobin and the other haemoproteins depends markedly on the cobalt ion oxidation state. It is possible that in the case of the cobalt phthalocyanine-modified peroxidase, oxidation of the cobalt ion of Co”L results in its incorporation into the protein in the proper orientation at the haem binding site. Carboxymethylation of the histidyl residues of the peroxidase apo-protein renders it completely inactive towards metal tetrasulphonated phthalocyanine, which suggests that, like in the case of haem, the histidine group constitutes the

1026

L. OSTROPOLSKA

phthalocyanine binding site. As is shown by molecular weight estimation, both phthalocyanine-modified peroxidases are monomers with molecular weights close to that of the native enzyme. Incorporation of the metal tetrasulphonated phthalocyanines into the haem crevice of the peroxidase protein virtually does not change its helical conformation, in spite of their larger size. On the other hand, the helical content of the examined model complexes is markedly lower than that of the native peroxidase, which points to their lower stability. 29-3’ Both phthalocyanine-modified peroxidases reduced with dithionite under anaerobic conditions are able to undergo reversible oxygen binding to give oxygenated compounds. The cobalt oxygen adduct, however, is unstable and undergoes slow auto-oxidation with degradation of the phthalocyanine ring. Both model complexes exhibit extremely low activity toward H202 and o-dianisidine, which indicates that they cannot fulfill the physiological function of the native peroxidase and cannot be regarded as its model in this respect. In conclusion, it can be stated that the haem crevice of the peroxidase closely fits to the natural prosthetic group, to give a conformation which is essential for the physiological function of the enzyme. REFERENCES 1. K. Kobayashi, M. Tamura and K. Hayoshi, J. Biol. Chem. 1980,255,2239. 2. G. McLondon and K. Sandberg, J. Biol. Chem. 1978, 253, 3913. 3. I. Morishima, S. Ogawa and T. Yonezawa, Biochim. Biophys. Acta 1978, 537, 293. 4. F. Basolo, B. M. Hoffman and I. A. Ibers, Accts Chem. Res. 1975, 8, 348. 5. I. Collman, Accts Chem. Res. 1977, 10, 265. 6. R. K. Dine110 and D. Dolphin, Biochem. Biophys. Res. Commun. 1978, 80, 698. 7. M. Tamura, T. Asakura and T. Yonetani, Biochim. Biophys. Acta 1972, 268, 292.

8. P. I. Ohlsson and K. G. Paul, Biochim. Biophys. Acta 1973,315,293. 9. T. Yonetani, H. R. Drott, J. S. Leigh Jr., G. H. Reed, M. R. Waterman and T. Asakura, J. Biol. Chem. 1970,245,2998. 10. M. R. Wang and B. H. Hoffman, J. Biof. Chem. 1977,252,6268. 11. M. Tamura, N. Skimidzu and K. Hayoshi, Biochem. Biophys. Res. Commun. 1977,75, 1029. 12. M. Tamura, T. Asakura and T. Yonetani, Biochim. Biophys. Acta 1973, 295,467. 13. R. K. Dine110 and D. Dolphin, Biochem. Biophys. Res. Commun. 1977,86, 190. 14. H. Przywarska-Boniecka, L. Trynda and E. Antonini, Eur. J. Biochem. 1975,52, 567. 15. L. Trynda, Znorg. Chim. Acta 1983,78, 229. and t. Ostropolska, J. 16. H. Przywarska-Boniecka Znorg. Biochem. 1982, 16, 183. and t. Ostropolska, J. 17. H. Przywarska-Boniecka Znorg. Biochem. 1984,20, 103. 18. L. M. Shannon, E. Kay and I. Y, Lew, J. Biol. Chem. 1966,241,2166. 19. T. Yonetani, J. Biol. Chem. 1967,242, 5008. 20. A. M. Crestfield, W. M. Stein and S. Moore, J. Biol. Chem. 1963,238,2413. 21. D. Vonderschmidt, K. Bernauer and S. Fallab, Helv. Chim. Acta 1965,48, 951. 22. P. Andrews, Biochem. J. 1965, %, 595. 23. M. J. Freehold, Enzymes, Enzyme Reagents and Related Biochemicals. Worthington Enzyme Manual, Worthington Biochemical Corp. (1972). 24. N. N. Ugarova and 0. V. Lebedeva, Biochimia 1978, 43, 1731. 25. H. Sigel, P. Waldemeir and B. Prijs, Znorg. Nucl. Chem. Mt. 1971,7, 161. 26. H. Przywarska-Boniecka and L. Trynda, Eur. J. Biochem. 1978,87,569. 27. B. M. Hoffman and D. H. Petering, Proc. Natf. Acad. Sci. USA 1970, 67, 637. 28. K. G. Welinder, FEBS Lett. 1973,30,243. 29. E. H. Strickland, E. Kay and L. M. Shannon, J. Biol. Chem. 1970,245, 1233. 30. E. H. Strickland, E. Kay, L. M. Shannon and J. Horowitz, J. Biol. Chem. 1968,243,3560. 31. P. I. Ohlsson, K. G. Paul and I. Sjijholm, J. Biol. Chem. 1972,252,8222.