Eur. J. Biochem. 87. 569-574 (1978)

Structural Studies of Iron and Cobalt Tetrasulfonated Phthalocyanine-Globin Complexes Helena PRZYWARSKA-BONIECKA and Liliana T R Y N D A Institute of Chemistry, University of Wrockiw (Received May 2, 1977jFebrual-y 20, 1978)

The structure of the complexes of iron and cobalt tetrasulfonated phthalocyanines with globin has been investigated by circular dichroism (CD), electron paramagnetic resonance (EPR) and polyacrylamide gel electrophoresis. Electrophoretic investigations and the molecular weight estimation indicates that the model complexes in the solutions are dimers. It is evident from the results of C D measurements that the incorporation of the iron or cobalt tetrasulfonated phthalocyanine into apohernoglobin significantly increases the helical structure of the protein and causes an appearance of the induced Soret and visible Cotton effects. Unlike methemoglobin, several discrete transition energies in the CD Soret band of Fe( 1II)L-globin are observed which suggests an inequivalence of the subunits within this complex. This suggestion is supported by EPR studies, which show that the iron atoms in Fe(1TI)L-globin are in two low electronic states. Electronic structures of the cobalt ions in Co(I1)L-globin and oxyCo( 1I)L-globin are similar to those of coboglobin and oxycoboglobin, respectively, as is proved by EPR results. On this basis we conclude that the oxygen adduct of Co(I1)L-globin can be described as a superoxide ion corrdinated to a formally cobaltic phthalocyanine compound. ln spite of the intensive research on the oxygenation process [l --41 the structure/function relation is still not known in detail. One of the ways of resolving this problem is to study the functional properties of the hemoglobin molecule after specific structural modification. Many investigations have been carried out in this field. It has been shown that substituting various unnatural iron porphyrins for the heme causes the significant differences in the conformation of the molecule [5,6]. Some attempts have also been made to incorporate the protoporphyrin of a metal other than iron into the heme crevice of apohemoglobin and apomyoglobin [7 - 1I]. Manganese and cobalt-substituted hemoglobins show many similarities with the original iron-containing proteins including, in the case of cobalt hemoglobins, the ability to bind oxygen reversibly. Manganese heinoglobin does not bind oxygen, but it undergoes conformational changes upon binding ligands, equivalent to those of hemoglobin. Our previous investigations [12] have shown that the interaction between cobaltous, ferrous and ferric tetrasulfonated phthalocyanines [Co(TI)L, Fe(II)L,

Fe(III)L] and globin results in the formation of the complexes whose properties resemble those of hemoglobin in many respects. These coniplexes can be obtained also by a displacement of heme in hemoglobin by phthalocyanine derivative, which suggests that phthalocyanine and porphyrin are bonded in a similar manner. Cobaltous and ferrous phthalocyanine-globins are able to combine reversibly with oxygen. The removing of oxygen from oxygenated phthalocyanineglobins is more difficult than in the case of hemoglobin, which points to the higher affinity of the former to oxygen. These compounds form also the complexes of higher stability with other additional ligands such as CO, NO, CN-, as well as organic bases. In order to provide additional data to the studies of the modified hemoglobins we have undcrtaken structural investigations of the new model complexes. The results of these investigations are described in this paper.

Ahhrevintio~is. L, tetrasulfonated phthalocyanine ligand, C32H12N8(S03Na)4; Fc( II)L, fcrrous tetrasulronated phthalocyanine: Fe(III)L, ferric tetrasulfonated phthalocyanine; Co(II)L, cobaltous tetrasulfonated phthalocyanine; C D . circular dichroism; EPR, electron paramagnetic rcsonance

Hemoglobin was prepared from fresh human blood by the method of Rossi-Fanelli and Antonini [13]. Globin was obtained using the method of RossiFanelli et al. [14].

MATERIALS AND METHODS

570

The preparation and purification of iron and cobalt tetrasulfonated phthalocyanines have been described earlier by Fallab et al. [15]. Stock solutions were obtained by weighing appropriate amounts of the solid and dissolving the latter in 100 ml 0.1 M phosphate buffer. The concentrations of hemoglobin and globin were determined spectrophotometrically. The absorption, A:t;1, of a 1-mg/ml solution of hemoglobin was taken as 0.85; A;;: for a 1-mgjml solution of globin was taken as 0.8. The concentrations of the complexes were determined from the molar absorption coefficients calculated for one subunit. To avoid errors in concentrations due to the adsorption of the free metal tetrasulfonated phthalocyanine impurity on the globin surface, molar absorption coefficients were determined for every new portion of preparation. Their average values are: Fe(lI1)L. globin 8641 = 3.7 x lo4 M-' cm-l, deoxyFe(I1)L . globin c678 = 3.6 x lo4 M-' cm-', deoxyCo(1I)L . globin &675 = 2.2 x lo4 M-' cm-'. The preparation of the globin complexes has been described [121' The reduced forms Of the 'Omplexes were prepared by addition of a few milligrams of sodium dithionite to their buffered solutions and removing the excess of the reductant on a Sephadex G-50 coiumn. Solid samples were obtained by lyophilization of the water solutions of the complexes. The stoichiometry of oxygen binding was determined manometrically. Oxygenated complexes were first converted into cyanmet derivatives using Drabkin solution: 0.05 mg K3[Fe(CN)6]/0.14 mg KH2P04. The amount of oxygen per subunit of Fe(I1)L-globin was found to be on average 0.86 mol, and per subunit of Co(1I)L-globin 0.90 mol. Circular Dichroism Measurements

The solutions of the iron and cobalt tetrasulfonated phthalocyanine-globins for circular dichroism (CD) measurements were prepared as follows. The reaction mixture containing globin and metal tetrasulfonated phthalocyanine in excess in aqueous solution was separated on Sephadex G-50 under equilibrium conditions at 4°C. Protein fractions were collected and lyophilized. Appropriate amounts of the lyophilisate was dissolved in Tris-HC1 buffer, pH 7 (0.02 M Tris, 0.01 M HCI, 0.1 M NaCI) or 0.02 M phosphate buffer, pH 7. Ultraviolet circular dichroism spectra were recorded using a model ORD/UV-5 Japan Spectropolarimeter, with C D attachment. Soret and visible C D spectra were measured in a model J-20 Japan Spectropolarimeter. Ellipticities are given per mole of bound metal tetrasulfonated phthalocyanine. Polyun.ylamidee Gel Electrophoresis The globin was incubated with appropriate metal tetrasulfonated phthalocyanine in phosphate buffer,

Iron and Cobalt Tetrasulfonated Phthalocyanine-Globin Complexes

pH 7, for 24 h. The reaction products were isolated by column chromatography and the protein fraction thus obtained was examinated by gel electrophoresis. Electrophoresis was performed in 7 % polyacrylamide gel in 0.052 M Tris-glycine buffer, pH 8.8, according to the procedure of Davis [16]. It was carried out at 4°C for 2-3 h. Gels with globin were then stained with amido black. Gels with metal tetrasulfonated phthalocyanine-globins were not stained because these complexes are intensively colored by themselves. Molecular Weight - Estimation Molecular weight estimations of the metal tetrasulfonated phthalocyanine-globins were carried out by gel-filtration on Sephadex G-75 columns according to the method of Andrews [17]. The following proteins were used as reference substances: cytochrome c ( M r 12400), myoglobin ( M , 17800), pepsin ( M , 35000), serum albumin ( M , 67000). In sedimentation experiments heterogeneity has been observed. Discrepancy between the results of the measurements points to the structural perturbations of the complexes, Electron Paramagnetic Rrsonunce ( EPR) Measuvrmeiz ts X-band EPR spectra of the polycrystalline complexes at liquid nitrogen temperature were performed with a Varian model JES-ME-3X Spectrometer. The microwave frequency was 9.27 GHz. The deoxyCo(I1)L-globin was prepared by two methods: (a) reduction of the oxyCo(I1)L-globin with dithionite in water solution and lyophilization of the sample in an argon atmosphere; (b) passing argon through the solution of the oxyCo(I1)L-globin for 6 h and then lyophilization in an argon atmosphere. The completion of the deoxygenation was estimated spectrophotometrically. RESULTS As shown in Fig. 1 on gel electrophoresis, all complexes of iron and cobalt tetrasulfonated phthalocyanines with globin give single bands exhibiting an electrophoretic mobility similar to that of globin. The bands of the iron derivatives are green and that of the cobaltous complex is blue (Fig. 1). Electrophoresis of oxyhemoglobin, compared to that of its mixture with Fe(1II)L-globin, after a 24-h incubation points to the dissociation of the tetraineric hemoglobin into dimers due to displacement of heme by Fe(II1)L. In Fig.2 are shown the results of gel filtration molecular weight determinations of the oxyCo(1I)Lglobin. The column was calibrated with cytochrome c, myoglobin, pepsin and serum albumin. The plot of elution volumes V against molecular weight shows that the molecular weight of the oxyCo(I1)L-globin

H. Priywarska-Boniecka and L. Trynda

571

140

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Fig. 2. Molecular. n.eigIi1 a/ o.xyC0(11) /,-glohin c ~ . c . / i r i ~ c ~ //ci ~j / ,gcd rhror?intograpliy on Sejiliudi~.xC-75. Plot of elution volume V against molecular weights of known proteins and model phthalocyanine complex. Coluinn size = 2.4 x 50 cm, equilibrated with 0.02 M potassium phosphate, pH 7.6

is about 38000 (Fig. 2). The approximate molecular weight of the Fe(I1)L-globin found in the same way is about 37000. These values are comparable with that of globin. Sedimentation experiments indicated structural changes effected by centrifugation. The stoichiometry of oxygen binding by Fe(I1)Lglobin and Co(I1)L-globin is found to be 1 mol oxygen/su buni t.

0

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Circular Dichmism Spectra The obtain the information regarding possible conformational changes of the protein due to the metal tetrasulfonated phthalocyanine binding, CD measurements of the model complexes were performed. The ultraviolet spectra of the iron and cobalt tetrasulfonated phthalocyanine globins, in comparison with the native globin, are shown in Fig. 3. Both phthalocyanine complexes, like globin, display negative bands at 222 nm and 209 nm. The intensities of these bands, however, are different. The ellipticities of Fe(II1)L-globin, oxyCo(I1)L-globin and native globin are at 222 nm: - 2.7 x lo6, - 3.4 x lo6 and - 2.25 x lo6 deg. cm2 dmol-', respectively; and at 209 n m : - 2.4 x lo6, - 2.15 x lo6 and - 1.75 x lo6 deg. cm2 dmol- ' respectively. OxyFe(T1)L-globin exhibits in this region a CD spectrum close to that of Fe(l1I)L . globin. The marked increase in the negative ellipticities represents an increase in the helix content of protein upon combination with metal tetrasulfonated phthalocyanine. If the globin has an estimated helix content of about 50 [18], Fe(II1)L-globin has

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about 60 and oxyCo(I1)L-globin about 74 '%; of the helix content. The near-ultraviolet CD spectrum of the globin contains a sinall positive band at 264 nin with 8 = 6.0 x lo3 and a larger positive one at 252 nm with 8 = 8.5 x lo3 deg. cm2 dmol-'. Fe(1II)L-globin, oxyFe(1I)L-globin as well as oxyCo(I1)L-globin have only

572

Iron and Cobalt Telrasulfonated Phthalocyanine-Globin Complexes

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Fig.4. In the Soret region (300-420nm) the oxy complexes of cobalt(I1) and iron(II), both exhibit positive Cotton effects at 350 nm with 8 values 6.0 x lo4 and 5.0 x lo4 deg. cm2 dmol-' respectively. The Soret C D spectrum of oxyCo(I1)L-globin is comparable to that of oxyhemoglobin. The oxyFe(I1)Lglobin C D spectrum is more asymmetrical and seems to be a multiple ellipticity band. The Soret C D spectrum of the Fe(II1)L-globin is more complicated. It consists of two positive bands at 350 nm and 370 nm with identical ellipticity 0 = 3.4 x lo4 deg. cm2 dniol-', besides one weaker positive band at 400 nm with 8 = 2.0 x lo4 and one negative band at 320 nm with 0 = - 2.0 x lo4 deg. cm2 dmol-'. The absorption spectrum of this complex shows only one band in the Soret region, at 340 nm. In the visible region Fe(II1)L . globin exhibits a negative C D band at 620 nni with 8 = - 2.7 x lo4 deg. cm2 dmol-', and another positive C D band at 640 nm with 0 = 2.5 x lo4 deg. cm2 dmol-'. This C D spectrum is associated with the absorption bands at 625 nm and 640 nm. Cobalt(I1) and iron(I1) oxy complexes show in the visible region negative Cotton effects at about 660 nm. The overall shapes of the spectra for both these complexes are very similar. They exhibit also identical ellipticities 8 = 4.0 x lo4 deg. cm2 dniol- I . Electron Paramagnetic Resonance Study

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one positive band in this region located at about 251 nm in the iron complexes and 255 nm in the cobaltous one. For all these complexes the ellipticity values are about 9.0 x lo4 deg. cm2 dmol-l. Similar results have been obtained with hemoglobin [19]. The Soret and visible C D spectra of iron and cobalt tetrasulfonated phthalocyanine-globins are shown in

The combination of Co(t1)L and Fe(II1)L with globin causes changes in the electronic structure of the central metal ions as has been shown by EPR investigations. Spin hamiltonian parameters for oxyCo(T1)L in MeOH are: gl= 2.004 (A?= 0.00092cm-'), g,, = 2.075 ( A $ ; = 0.00149 cm-'). For deoxyCo(I1)L in MeOH these parameters are: gl= 2.27 ( A T = 0.0009 cm-I), g,, = 2.065 (A"?= 0.00107 cm-') [18]. The EPR spectrum of the solid oxygenated Co(1I)L . globin at the temperature of liquid nitrogen is presented in Fig. 5a. It is characterized by five overlapping resonance lines appearing at g = 2.39, 2.25, about 2.07, 2.005 and 1.99 (Fig. 5). The line at g z 2.07 is rather broad suggesting a possible superposition of two EPR signals. Nevertheless, it shows an eight-line hyperfine structure with a coupling constant A = 0.00146 cm-'. Reduction of the oxyCo(I1)L-globin by dithionite or deoxygenation by passing argon through its water solution for 6 h produces the characteristic changes in its EPR spectrum (Fig. 5 b, c). The weak signal at g = 2.39 becomes stronger and that at g = 1.99 markedly decreases. Moreover, the significant lowering in the intensity of the signal in the vicinity of g = 2.07 is observed. These experiments allow us to ascribe the EPR line at g = 2.39 to the deoxyCo(I1)L-globin and two other lines at g z 2.07 and 1.99 to the oxyCo(I1)L-

573

H. Przywarska-Boniecka and L. Tryiida

globin. The spin hamiltonian parameters for these phthalocyanine complexes are similar to the corresponding parameters of deoxy and oxycoboglobin as well as to vitamin Blz parameters in its deoxy and oxy forms. According to this analogy the g = 2.39 represents the perpendicular branch of the deoxyCo(I1)Lglobin. The parallel component of this spectrum occurs at g about 2.06. It is broad, and an accurate value of g,,cannot be defined. For the oxyCo(I1)L-globin the perpendicular line should be assigned to gL = 1.99 and the parallel one to gll z 2.07. The presence of 8 hyperfine lines in the EPR spectrum of the oxyCo(1I)L . globin is probably due to a 59Co nucleus and an unpaired electron interaction. It indicates that the unpaired electron is not far distant from the cobalt atom. The interaction with oxygen, however, lowers the electron density on cobalt and is responsible for the smaller value of g1 as compared to g,,. These results suggest that we should consider this complex as a paramagnetic cobalt(II1) complex with the superoxide anion as an additional ligand. EPR signals at g = 2.25 and 2.005 are unaffected by argonating the oxyCo(I1)L-globin solution. They are believed to be due to aggregated Co(1I)L presumably adsorbed to the protein. Reduction by dithionite bring about the failure of the line at g = 2.25 and give rise to an additional broad signal in the vicinity of g = 2.13, probably resulting from a Co(1I)L reduction product [20]. Electron paramagnetic resonance examination of Fe(II1)L shows that this complex in aqueous solution exhibits no detectable EPR signal. In these conditions Fe(1II)L exists mainly as a dimer and there is evidently sufficient coupling to wipe the signal out. In diniethylsulphoxide, however, a narrow signal at gvalues = 2.003, 1.99 and 1.97 appears, which indicates an octahedral low-spin state of the iron atom with slight rhombic distortion. No superhyperfine structure is observed in this spectrum. The ferric tetrasulfonated phthalocyanine-globin displays at liquid nitrogen temperature an EPR spectrum which consists of two superimposed low-spin signals (Fig.6). One of them is centered around g = 2.0. It exhibits rhombic symmetry with gl= 2.03, g,,, = 2.003, g,, = 1.99. In the perpendicular component a superhyperfine structure is observed. Its resolution is poor. The existence of the superhyperfine structure in the phthalocyanine complex is unclear. Another EPR signal, centered at a lower-field region, is broader and shows slightly recognizable anisotropy with gl= 2.46 and

i

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H

Fig. 6 . E P R spectrum nitrogen temperature

UJ'

pol~crystallineF e ( l l 1 )L-globin (it liquid

been confirmed by Mossbauer resonance experiments (D. S. Kulgawczuk and H. Hrynkiewicz, unpublished results). CONCLUSION In our previous work [12] we have shown that it is possible to obtain reconstituted hemoglobins with cobalt and iron tetrasulfonated phthalocyanines in the place of heme. The results presented in this paper allow us to draw some conclusions about the structure of these compounds. Electrophoresis patterns in Fig. 1 demonstrate that the reconstituted hemoglobins are dimers. They move in a manner similar to that of globin. Moreover, the displacement of the heme in the oxyhemoglobin by metal tetrasulfonated phthalocyanine results in the dissociation of the hemoglobin tetramer into dimers. Further evidence of the dimeric structure of the new complexes are their molecular weights (37 000 - 38 000) which are similar to that of globin (40000). In spite of their dimeric structure Co(I1)L-globin and Fe(I1)Lglobin are able to bind oxygen reversibly. The stoichiometry of this reaction is 1 mol oxygen/subunit of the complex. The combination of globin with metal tetrasulfonated phthalocyanines virtually does not change the shape of the protein CD spectra in the ultraviolet but it results in a significant increase of the negative ellipticity at 222 nm and 209 nm. That points to the stabilization of the secondary structure of the protein by the phthalocyanine complex.

574

H. Przywarska-Boniecka and L. Trynda : Iron and Cobalt Tetrasulfonated Phthalocyanine-Glohin Complexes

The near-ultraviolet Cotton effects resemble that of hemoglobin. In the Soret and visible part, significant differences between the CD spectra of the complexes examined are observed. Oxygenated Fe(I1)L-globin and Co(I1)L-globin exhibit simple CD spectra in this region, comparable with that of oxyhemoglobin. The C D spectrum of Fe(II1)L-globin is more complex and demonstrates several discrete transition energies within the simple Soret absorption band. Empirical studies of the Soret circular dichroism shows a sensitivity to events of a quaternary nature [21,22]. Calculations by Hsu and Woody account for the Soret circular dichroism of hemoprotein as the result of coupled oscillator interaction between heme transitions and allow n - U * transitions in nearby aromatic amino acids [23]. The splittings of the Soret CD band of Fe(II1)L-globin result probably from a subunit inequivalence. This suggestion is supported by EPR results. The EPR spectrum of the Fe(II1)L-globin demonstrates the existence of two different low-spin ferric ions in the complex. The low spin of the iron ions in Fe( 1II)L-globin is not surprising. Phthalocyanines are high-ligand-field molecules and all their metal complexes are of the low-spin type. The redox data provide evidence that phthalocyanines are exceptionally good n acceptors [24]. Their electronic state and redox characteristics exhibit greater sensitivity towards the environment and axial ligation than those of the porphyrins. The porphyrin ligand field strength is relatively low, which is in agreement with the minimal capability of the metal porphyrins and their tendency to occur in the high-spin state, as in the case of the natural methemoglobin. The electronic structure of the Co(I1)L-globin central metal ions resembles that of coboglobin. The EPR spectrum of this complex is typical of low-spin cobalt(I1) complexes containing a single axially coordinated base. Superhyperfine splittings from the nitrogen of the proximal histidine cannot be identified because of a poor resolution. Oxygenation of Co(1I)Lglobin produces a new low-spin signal in its EPR spectrum. Its spin hamiltonian parameters are similar to those of oxycoboglobin and other oxygenated cobalt(I1) complexes [25]. We conclude that oxygenation results in the formation of an oxygen adduct of Co(1I)L-globin which, like the other oxygenated cobalt complexes, can be described as a superoxide ion coordinated to a formally cobaltic phthalocyanine compound. Oxygenated Co(I1)L-globin is stable. It does not undergo further oxidation to a marked degree. No changes detectable by EPR are observed in the EPR spectrum of a given sample, at least over a period of several weeks. On

the contrary, cobalt(I1) tetrasulfonated phthalocyanine with axially coordinated imidazole is stable only in anaerobic conditions [26]. This fact suggests that the oxidation state of the cobalt ion in Co(I1)L is stabilized by coordination of this complex with globin. EPR spectra of the Co(I1)L-globin upon oxygenation always display the presence of some of the deoxy form. The reason of this fact cannot be explained without detailed kinetic and thermodynamic studies of the Co(I1)L-globin-O2 system.

REFERENCES 1. Antonini, E. & Brunori, M . (1971) HemcJglObin and Myoglobin in Their Reu(,tioii.( with Li,qands, pp. 381 -415, North Hol-

land, Amsterdam. 2. Perutz, M. F. (1972) Nature (Lond.) 237, 495-499. 3. Baldwin, J. M . (1975) Prog. Biophys. MoE. BioE. 29, 225-320. 4. Fung, L. W. M., Minton, A. P. & Ho, C. (1976) Proc. Nut1 Acad. Sci. U.S.A. 73, 1581-1585. 5. Gibson, Q. H. & Antonini, E. (1963) J . Biol. Chem. 238, 1384-1388. 6. Antonini, E., Brunori, M., Caputo, A , , Chiancone, E., RossiFanelli, A. & Wyman, J. (1964) Biochim. Biophys. Acta, 79, 284- 292. 7. Andres, S. F. & Atassi, M. Z. (1970) Biochemistry, 9, 22682275. 8. Yonetani, T., Drott, H. R., Leigh, J. S., Jr, Reed, G. H., Waterman, M. R. & Asakura, T. (1970) J . BioE. Ckem. 245,29983003. 9. Fahry, T. L., Simo, C. & Javaherian, K. (1969) Biochim. Biophys. Acta, 160, 118 - 122. 10. O’Hagen, J. E. (1961) Hematin Enzymes, p. 173, Pergamon Press, London. 11. Hoffman, B. M. & Petering, D. H. (1970) Proc. Natl Acad. Sci. U . S . A . 67, 637-643. 12. Przywarska-Boniecka, H., Trynda, L. & Antonini, E. (1975) Eur. J . Biochem. 52, 567-573. 13. Rossi-Fanelli, A. & Antonini, E. (1955) Arch. Biochem. Bioplays. 58, 498 - 499. 14. Rossi-Fanelli, A,, Antonini, E. & Caputo, A. (1958) Biochim. Biophys. Acta, 30, 608 - 615. 15. Vonderschmitt, D., Bernauer, K. & Fallab, S. (1965) Helv. Chim. Acta, 48, 951-954. 16. Davis, B. J. (1964) Ann. N . Y . Acad. Sci. 121, 404. 17. Andrews, P. (1964) Biochem. J . 91, 222-233. 18. Abel, E. W., Pratt, J. M. & Whelan, R. (1971) Chent. Commun. 449-450. 19. Schrauzer, G . N. & Lee, L. P. (1970) 1. Am. Chem. Soc. 92, 1551 - 1557. 20. Rollmann, L. D. & Iwamoto, R. T. (1968) J . Am. Chem. Soc. 90, 1455 - 1462. 21. Geraci, G. & Li, T. K. (1969) Biochemistry, 8, 1848-1856. 22 Sugita, Y., Nagai, M. & Yoneyama, Y. (1971) J . Biol. Clienz, 246, 383 - 388. 23 Hsu, M. C. & Woody, R. W. (1971) J . A m . Chrm. Soc. 93, 3515- 3525. 24 Lever, A . B. P. & Wilshire, J. P. (1976) Can. J . Chem. 54, 2514- 2516. 25 Walker, F. A. (1970) J . Am. Chern. Soc. 92,4235-4743. 26 Rollmann, L. D. & Chan, S. I. (1971) Inorg. Chen7. 10, 19781982.

H. Przywarska-Boniecka and L. Trynda, Instytut Chemii, Uniwersytet Wroclawski imienia Bolesbawa Bieruta, Ulica Fryderyka Joliot-Curie 14, PL-50-383 Wroctaw, Poland