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Complexes of iron and cobalt tetrasulfonated phthalocyanines with apocytochrome c
Complexes of Iron and Cobalt Tetrastionated Phthdocyanines with Apocytochrome c Helena Przywarska-Boniecka and Lucja Ostropolska Insiitute of Chemistry, University of Wrockzw, Poland
ABSTRACT ArtificiaI cytochromes c have been prepared with Fe(III) and Co(ilI) tetrasulfonated phthalocyanines in place of heme. Their structure and properties have been lnvestigated by difference spectroscopy, CD, epr, electrophoresis, molecuIar weight estimation, and potentiometric measzements. The visiile absorption spectra show the main peak at 650 mn for the iron compound 685 nm for the cobalt one. It is shown by CD experiments that incorporation of Fe(III)L or Co(III)L into apocytochrome c markedly increases helical content of the protein. Its conformation is, however, significantly altered as compared with the native cytochrome c. The epr and spectroscopic data show that the iron and cobalt phthalocyanine models represent the low spin species with the metal ions in trivalent state. Eiectrophoresis and molecular weight estimation indicate these complexes to be monomers_ Both phthalocyanine complexes have not affinity for additional ligands characteristic for hemoglobin_ They react, however, with CO, NO, and CN- whin they are reduced with dithionite. Moreover. Co(II)L-apocyt c is able to combine with oxygen suggesting a structural feature in common with the oxygen-carrying heme proteins. Iron complex in the same conditions is oxidized directly to the ferric state. The half-reduction potentials of Fe(III)L-apocyt c and Co(III)L-apocyt c are +374 mV and +320 mV, respectively. These complexes are reduced by cytochrome c and cytochrome c reductase (cytochrome bq).
Abbreviations L, tetranrlfonated phthalocyanine ligand [C32H12N&03Na)4] ; Fe(III)L, iron(M) tetrasuIfonated phthaiocyaniue; Co(III)L, cobalt(III) tetrasulfonated phthalocyanine; apocyt c, apocytochrome c.
INTRODUCTION Specific modifi~tions of the native metalioenzymes have been accepted as one of the most usefui methods to study function-structux relationships of these compounds. Address reprint requests to Docdr hab_ Helena Boniecka, htytut ul.F.JoIiotCurie 14, SO-383 Wroolaw_ Jo-~of~norg~ic~~~~~m~~16,183-199 0 Elsevier Science Pub!ishiug Cp., Inc_, 1982
52 VandzrbiIt Ave., yew York, NY 10017
Chemii Uniwersytetu Wrodawskiego,
(1981)
183 0162-0134/82/030183-1752.75
184
H. Przywarska-Boniecka and L. Ostropolska
Recent studies have shown thatit is rassible to modify hemoglobins. myoglobins, [l-5], and cytochrorue c [cl I] by inco.porating the protoporphyrin of metal other than iron into the heme crevice of their apoproteiris. Investigations of these metal substituted derivatives gave valuable infomations about mechanism of the allosteric transition in hemoglobin and shed light on the requirements for electron transfer processes in CJtochrome systems [ 123. Cobalt derivatives allowed epr studies which are not possible fclr diamagnetic ferrous species. The compounds which closely resemble iron porphyrin are iron and cobalt pi~thaic~ya-ines as well as their water soluble tetrasulfonatedderivatives [ 131. Earlier we have shown that substitution of heme in hemoglobin by metal tetrasulfonated phthaiocvanines gives dimeric complexes v/hose properties resemble those of the native species [14, 151. Combination of iron phtha!ocyanine derivative with apocatalase, however, gives denatured species [ 163_ We now report the re~lts concerning the preparation, structure, and properties of artificial cytocbromes obtained by incorporation of iron and cobalt tetrasulfonated phthalocyanines into the heme binding site of a-3 tochrome c _
Pig heart cytxhrome cbromato@fic~y
c was purchad
from Biomed (Krakow) and purified
[ 17 J.
Apocyt~~trome c was prepared by modification OFthe silver sulfate method [IS]specifically, 160 mg of Ag$O, in IS ml of water am! 1 6 ml of acetic acid were added to 100 mg of cytochrome c disolved in 2 ml of water. The solution was incubated in the dark for 4 hr a& 44°C zd was then centritiged to remove heme aggregates. The apoc:ytocLmme t was separated from the heme and precipitatedby addition of 15 ml of cold
Tetrasulfonated Phthalocyanines and -4pocytochrome c
185
The synthesis of the metal phthalocyanine substituted cytochmme c involves the incubation of apocytochrome c with twice excess of the metal tetrasulfonated phthalocyanine in 0.2 M acetic acid for 5 days at 4°C. The reaction mixture was separated by gel filtration on Sephadex G-50 and the fracticns were identified by absorption spectroscopy at 280 and 650 or 685 run, respectively, for the iron and cobalt derivatives. The bluegreen protein fraction were iyophilized. The reduced forms of the complexes were prepared by addition of a few milligrams of sodium dithionite to the solutions and removing the excess of the reductanton a Sephadex G-50 column in argon atmosphere. The concentrations of the complexes were determined from the molar absorption coefficients. To avoid errors in concentrations due to the adsorption of the free-metal phtbalocyanine impurityon the protein surface, molar absorption coefficients were determined for every portion of preparation. Their average values were thus: Fe(III)L-apocyt c, em = 2.6 x 101 M-l cm-‘; Co(JII)L-apocyt c, esss3.75 x 101M-r cm-*, Fe(II)L-apocyt c, ~~ = 3.16 x l(r M-* cm-r; Co(II)L-apocyt c, r,, = 2.9 x l(r M-r cm-r _ Hemin was obtained from Koch-Light Chemical Co. The combination of the metal phthakyanine substitutedcqtochromes c with nitric oxide was carried out anaerobically. Purified argon was passed through the solution for 20 mitt and then was replaced with nitric oxide by passing NO through the solution for a further 20 min. Evaporated water was completed in anaerobic conditions at 4°C. The sample was allowed to stand for 24 hr before use in spectroscopic and epr experiments.
Absorption
Spectroscopy
Absorption and difference spectra were performed on a Car-r 15 recording spectrometer with a cell compartment thermostatically controiled at 15°C or on Specord recording spectrophotometer. The measurementswere made under equilibnzm conditions. Reaction with Hemin The reaction of Fe(IJJ)L-apocyt c with hemin was carried out in molar radioof complex to hemin 1:4. The reaction mixture was incubated at 4”1=in the dark for 14 days and then separated chromatographically. It was applied to a column of Sephadex G-50 equilibrated with O-05 M acetate buffer pH 5.8. Elution was carried out with the same buffer at a flow rate of 25 ml/hr. Three-milliliter fractions were collected and examined spectrophotometrically. The gel filtration profile was made for A,, A,,, , and A,, _ Cinrillar Dichroism Measurements Circular dichroism (CD) spectra were recorded using a model ORD/uv-5 Japan Spectropolatimeter with CD attachment. The solutions were prepared by dissclving appropriate amounts of lyophilizated preparation in acetate buffer, pH 5.8, or in 50% cbloroetllanol. EIktl7lllP :aramagnetic Resonance (epr) X-band epr spectra of the complexes in buffered solutions were performed at liquid nitrogen temperaturewith Varian model JES-ME-3X spectrometer.The solutions under
186
H. Przywarska-Boniecka and L. Ostropolska
investigation in these conditions become fi-czen and, experimentally, they behave as glassy media. The microwave frequency was 9.27 GHz. The epr spectra of the reduced complexes or their NO derivatives were made in anaerobic conditions. PoIy-crylamide Gel F.&ctrophoresis Electrophoretic studies were carried out according to the method described earlier [ 151.
Moh=cdar Weight Estimation Molecular weights of the model compleres wete determined by gel filtration on Sephadex G-75 column according to the method of Andrews (see Stellwagen et al [ 181). I he following proteins were used as reference substances: insulin (MW 6,000), cytochrome c (Mw 12,400), lyzozyme (MW 14,000), myoglobin (MW 17,8CK!),pepsin (MW 35,000), and serum albumin (MW 67,000).
OxidatiorkReduction
Potentials
The oxidation+eduction potentials of the model complexes were studied by mixture method [22] which consists of the measurements of the potential of mixtures of the oxmixed and reduced forms of the examined compound in known proportion. The fully reduced preparations were obtained according to the method described earlier !8]_ The full oxidation was performed by addition of stoichiometric amounfj of K3 [Fe(CN),] to tie soiutions of the examined complexes [5 x 10-j M] and separation of the oxidation pro&co on a Sephadex G-50 coiumn equilibrated with 0.005 M phosphate buffer, pH 7, and 0.2 M KC1 _ The molar concentrations of the complexes were determiner; from the molar absorption coefficients. Oxidatio-reduction pc:entials were determmed in a titration chamber similar to those employed by Clark [23]. it was fitted for argon purge, platinum flag electrode, and saturated calo.mel electrode with a KCl-agar salt bridge. Voltages were measured with type V-627 potentiometer. Absorbance was followed by Cary 15 spectrometer. A mixture of mediators was used f0.5 PM] to facilitate the equilibration of the oxidize&reduced forms. They are Ks[Fe(CNX], methylene blue, thionine, and to!uene blue. A known amount of fully oxidized complex in buffered solution was titrated in argon atmosphere with increasing amounts of its reduced form,
Assay
Enzymatic
Enzymic reduction of Fe(III)L-apocyt c and Co(iII)L-apocyt c was assayed with system involving mitochondrial cytocbrome 5 reductase and NADH as a substrate, disso,ved in 0.1 M phosphate buffer, pH 7, made 0.1% in Triton X-100. All solutions were deoxygenateti by argon purging over 2 hr. The assays were performed by injection anaerobically appropriate quantiries of NADH and cytocbrome c reductase (bc, = [2.2 x lo-“, MJ, complexess = 11.2 x IO- 5, Ml). The reaction run was followed spect.rophGtometrically.
RESULTS Artificial cytochromes c cannot be obtained by a displacement of heme in cytochrome c with metal tetrasulfonated phthalocyanines. Instead, phthalocyanine derivatives are
Tetrasulfonated
Phthalocyanines
and Apocytochrome
c
187
reduced with cytochrome c as is observed in the difference spectra presented in Figure 1. Characteristic absorption peak for iron tetrasulfonated phthalocyanine in water solution is at 635 run. Water solutions of cobaIt(II) tetrasulfonated phthalocyanine contain an equilibrium of monomer and dimer with characteristic absorptions at 668 and 625 run. These peaks disappear when cytochrome c is added to such solutions and new ones arise at the same time, at 670 nm for the iron complex and at 698 and 740 nm for the cobalt one. The position of the new bands points to the reduction of the phthalocyanine complexes 124,251. Cytocbrome c, on the other hand, is oxidized to its ferric form as is demonstrated by disappearance of its band at 424 nm and the arise of another one at 408 mn which is characteristic for the ferric cymchrome c. I’nteraction of Iron and Cobalt Phtbalocyanine Complexes (Fe(IIZ)L and
Co(Il)L) with Apocytochrome
c
The reaction between metal tetrasuifonated
phthaiocyanines and apocytochrome c was proved by the difference spectroscopy experimenrs. Visible difference spectrum of Fe(IIi)L-apocytochrome c system shows two positive peaks at 650 and 695 nm, and a negative one at 635 mn. This points to the formation of the complex between phthalocyanine derivative and apocyt~hrome c (Fig. 2). Visible difference spechurn of Co(II)L-apocytochrome c system exhibits a positive peak at 685 nm and two negative peaks at 625 and (68 nm. Spectrophotometric titration data indicate the formation of the metal-phthalocyanine-apocytmk-ome c cor;pIexes at the mokir ratio of the reactants 111. Incubation of Fe(III)L-apocyt c with hemin in acetate buffer pH 5.8 for 2 weeks leads to the displacement of Fe(III)L by hemin and reconstitution of cytochrome c. The results of the chromatografic separation of the reaction mixture are presented in Figure 3. The eiution profile exhibits three well-resolved peaks at the positions of 72(I), 84(II), and 162 ml(III). Two leading fractions are proteins with charac+tic absorptions at 650(I) and 413 nm(II). Fraction I which appears at the solvent front is blue and has been identified as Fe(III)I-apocyt c. Fraction II exhibits abmrption spectrum corresponding to that of cytochmme c. The lower moIecular weight peak represents the bIue fraction (III) with characteristic absorption, a:. 635 nm. It has been identified as Fe(III)L. The unreacted hemin remains on the coiumn graduaiIy spreading from the origin. The affinity of the model complexes for additional ligands were studied spectroscopically. In experimental conditions, they do not have the ability to coordinate with imidazole, CO, NO, and CN- _ They react, however, with these iigands when they are reduced, this is demonsL;ated by the increase of intensity of their main absorption bands with minimal batochromic shift. The complex of cobalt tetrasulfonated phthakyanine with apocytochrome c virtually has not the ability to reverse the oxygen binding, suggesting that an insertion of Co(II)L into apoprotein in aerobic conditions leads to oxidation of cobalt ion to its trivalent state. The phtbalocyanine substituted cytochromes c are reduced with dithionite as is shown by characteristic changes in their absorption spectra iFis_ 4). Figure 4A presents visible difference specaum of Fe(III)capocyt c reduced with dithionite against the same complex -unreduced. The reduced form exhibits two absorption bands at 680 and 608 NTL This skxtrurn rrrzverts into that of Fe(III)L-apocyt c in an air atmosphere.
The reductton of Co(III)L-apocyt c with dithionite in argon results in a disappearance of thy band at 685 nm and an arise of a new one at 638 nm (Fig. 4B). On exposure to
) mixturesagainstthe snmeaolutlons FIGURE 1. Differencespectra of Fe(lll)L t ferrous cytochrome c (-- ) nnd Co(ll)L t ferrous cytochrome c 6-v unmixed.R(III)L = Co(II)L = (8.6 x 10-s Mj, ferrous cytochromec = [5,54 X 10-e M].
Ak
Tetrasuifonated Phthalocyaninas and Apocytochrome
c
189
(nm 1
FIGURE 2. Difference spectra of ke(III)L + apocytochrome c (-) and Co(II)L + apocytochrome mixtures against the same solutions unmixed: Fe(III)L = Co(II)L = [2.0 x 104 M] , apoc (---) cytochromec= [LOX 10-5&f].
oxygen, reconstitution of the band at 685 nm is observed. It is, however, more intense than that of the original complex. The band at 638 nm disappears at the same time. When bubbled with argon, the oxygenated solution zailses a gradual decrease of the absorption at 685 run. Prolonged bubbling with oxygen leads to an irreversible oxidation of the cobalt ion. The spectrophotometric experiments indicate that Co(III)L-apocyt c reduced with dithionite can combine reversibly with oxygen, giving an oxygenated intermediate_ Similar results are obtained with an ascorbate as the reductant, but in this case the reaction is slower than that with dithionite.
!Sctmphoresis
and Molemhr
Weight Estimation
Polyacrylamide gel electrophoresis patterns show that the electrophoretic mobilities of both model complexes are identical with that of the native enzyme. The results of the molecular weight estimation of Fe(III)L-apoc: t c and Co(III)Lapocyt c show that the molecular weight of the iron complex is approximately 13,000 and that of the cobalt complex is 12,!JOO.These values are comparable with that of cytochrome c.
circuIarDichroismspectM The conformationaL properties of the model complexes were examined by CD technique in acetate buffer, pH 5.8. and in 50% 2chloroethanol. For comparative purposes, CD spectra of apocytochrome c and native cytochrome c were performed at the same
FIGURE 3. The gel fdtrction profile of the reaction products of F’c(lll)L-apocytc with hemin in 0,05 M acetate buffer, pH 5.8, on a column (2 x 85) of ScphadexG-50at 25 ml/hr, 4°C I,&~o; II,,4413; ill,A6g6,
Frraction number 9
6. r
Tetrasulfonated Phthalocyanines and Apocytochrome c ,
191
FIGIiRE 4. Differencespectraof (A) Fe(IIDL-apocytc + ??a&$4
and (B) Co(III)L-apocyt c + Na2S204 mixtures against the same solutions of the complexes without dithionite, in argon (-) and in aerobic conditions (---)_ Fe(iJI)L-apocyt c = 12.4 x 10-5 Ml, Co
conditior;s. In acetatebuffer, apocytochrome c exhibits a large negative maximum near 200 run accompanied by negative infkction near 225 MI. This patternis indicative of a predominantly unordered conformation. In 2-chloroethanoI. however, CD spectrumof apocytochrome c is characterizd by two intense negative bands at about 210 and 223 nm. Both spectra are in agreement with those previously reported 1261. Native cytocbrome c displays a strong Cotton effects at 210 and 223 nm in acetate buffer as welI as in 2-chloroethanol_ Ellipticity vaiues expressed in deg cm* dmol-’ are -7.9 x 105 in acetate buffer (Fig. 5A), Btlo = - 1.85 X 106, 8t,0=-6~105,8tu= 9 tu = -2.25 X 106in 2-chlomethanole (Fig. 5b& The phthalocyaninemodel complexes exhibit negative uv Cotton effects at 210 and 223 nm in both solvents. The elliptic@ values, however, are significantly lower than those of cytochmme c. In acetate buffer they are & = -3.0 x 105,em = -2.7 x 105for the cobahic complex, & = -3.4 X
192
H. Przywarska-Boniecka
and L. Ostropolska
-
300 WWeiE!!gh
x)
210
220
230
W avelengh
240
(nm)
-@
(nm) -240
Fe(III)LFIGURE 5. Ultraviolet CD spectra of cytochrome c (-- ), apocytochmme c (--), apocyt c (------). and Co(III)L-apocyt c (-----;) in (A) 0.2 M acetate buffer, pH 5.8, and (J3)in 2-chhoethanok. (Cl CD spectraof the model compiexesin acetatebuffer io the Soret region
Tetrasulfcmated Phthdlocyznines and Apocytochrome
c
193
em=
-3.2 X f(Y for the ferric one. The elliptic@ values for the ferric compIex in 105, 50% 2chloroethanol are &,,, = - 1.65 x 106, t& = 1.85 x 106. All ellipticity values are expressed in deg cm’ dmol-1 . In the Soret regian (300420 nm) the model complexes in acetate buffer both display induced Cotton effects with two positive CD bands of identical intensity (Fig. 5C). They are observed at 315 and 333 nm in CD spectrum of Co(iII)L-apocytochrome c (0 = 2.1 x 104 deg cm’ dmol -1) and at 335 and 347 run in that of Fe(III)Capocyt c (e = 2.1 x 1oQdeg cm* dmol -I)_ The absorption spectra of these complexes show in this region one broad band which appears at 325 nm for the cobalt complex and at 342 nm for the iron one. Electron ParamagneticResonance Study The epr studies of Fe(lIT)L-apocyt c at pH 5.8, 77”K, shows distinct broadened low spin signal with a central g value of 2.106 (Fig. 6). It has no observable hypetfine splitting- Asymmetric shape of this signal could suggest superpositionof the signals of two low spin species. Afterzduction with dithionite, epr spectrum disappears due to low spin Fe(iI)L-apocyt c. Nitric oxide derivative of Fe(II)L-apocyt c exhibits an intense epr spectrum with the central resonance signal at g = 2.084. The splitting from the hypctfine interaction with nitrogen nucleus of the NO Iigand is not observed in this spectrum indicating that the unpaired electron of NO spends a considerable fractionof time in the iron orbitals. The epr spectrum of the cobalt phthalocyanine model of cytochrome c at pH 5.8 is more complicated (Fig. 7A). It consists of two broad, weak signals at the low and high field regions. The weaker low field signal exhibits g = 3.53. The high field signal is more intense and shows partially resolved hyperfine structure. Its spin Hamiltonian parametersareg = 2.31,g = 2.03, A = 39G, A = 91 G. Additionofdithionite to the examined solution produces the characteristic change of its epr spectrum (Fig. 7B). The intensity of the low spin signal markedly increases what points to the reduction of the majority of the original preparation. Beside the hyperfine structure partially resolved, supe .hyperfiie lines from one nitrogen of the axial ligand are observedThe epr study results suggest that the main species of the preparation constitutes Co(III) complex. The low field minority component of epr spectrum is probably due to impurity of Co(II)L coordinated to some unspecific protein residue.
oxidation-Reduction
Potentials
Figure 8 shows oxidatiorr-reduction equilibrium curves of the complexes of iron and cobalt tetrasulfonated phthalocyanines with apocytochrome c at 25°C as obtained by the mixture method. Methylene blue was used as mediator in these experiments. The values of &Xl., given by the oxidizing titrations are +375 and t320 mV for iron and cobalt complexes, respectively. I&se values are reproduced in the reducing titrations and in the presence of the mixture of the following mediators: K,Fe
H. Przywarska-Boniecka
194
and L. Ostropolska
>
3m FIGURE 6. X-band epr spectn of (A) Fe(iII)L-apocyt buffer, pH 5.8, at liquid nitrogen temperature.
__3mo
3300
H
c and Q) its NO derivative in 0.2 M acette
reaction mixtures (Fig. 9). They show a rapid decree of the band characteristicfor the oxidized form of the model complexes and an appearanceof thatcorresponding to their reduced form. Simuhaxously, growth of the band at 424 MI characteristicfor the oxidized cytochrome reductase is observed. The e xamhed complexes are reduced ako Sy the native qtochrome c; this is in agreement with their redox potentials (A?& of cytochrome c is t260 mV).
Tetrasulfonated
PhthalocyaGnes
and Apocytochrome
c
195
FIGURE 7. X-band epr spectraof Co(III)L-apocyt tin 0.2 M acetate buffer, pH 5.8, (Al in original form, (Is) rexiucedwith dithionite.
H. Przywarska-Boniecka and L. Ostropolska
196
FIGURE 8_Oxidatio=-reduction titration of Fe(lII)L-apocyt c ( -) and Co
CONCLU§ION The interaction between the iron and cobalt tetrasulfonated phthalocyanines and apocytochrome c leads to the formation of artificial cytochromes c with the iron and robalr phtbalocyanine derivatives in place of protoheme. The visible spectra of these complexes are characteriz& by a main intensepeak at 650 mn for the ferric compound and at 685 nm for the cobalt one. In addition, the ferric complex exhibits a weaker band at 695 nm. Tbe 695 nm band in the ferric cytoehrome c is considered to he strong evidence for the ligr tion of methicnine-80. However, it is relatively intensein Fe(III)Lapoeyt c and cannot be assigned to the Fe-S bond without additional proofs. In contrary to hemoglobin, heme in cytochrome c cannot be displaced by the metal phtbalocyanines. It is covalently bonded to the protein by strong thioether bridges between the porphyrin ring and two cysteine residues in the peptide chain. Phthaloeyanines do not have the capability to form such bonds. Instead, metal tetrasulfonatedphthalocyanines are removed from the model complexes by hemin with reconstitution of the ferric cytochrome c. This fact suggests that the metal phtbalocyanine binding site in apocytochrome c is the same as that of hemin in cytochrome cc _ The identification of the axial ligands in the examined complexes is uncertain at present. They are not displaced by such molecules as CO, NO, cyanide, and imidazole; they then seem to he strong field ligands such as histidine, lysine, or me&ion&. In the reduced form, however, both phthalocyaninecomplexes can readily bind additional Iigands. It is assumed thatthe reactionof the examined complexes with ditbionite or ascotbate causes conformational change that leads to the loosening of the
Tetrasulfonated
Phthalocyaninx
and Apocytochrome
axJoq1osq\d
197
c
0
H. Przywarska-Boniecka and L. Ostropolska
198
heme crevice relative to the original species_As a result, a substitutionof an axial ligand by another from outside is easily made. The iron and cobalt phthalocyanine models both represent the low spin species. The epr spectra indicate that the cobalt ion in the model complex appears mainly in the trivalent state and reduction with dithionite yields a Co(R) state. Some portion of the original preparationexists as a six-coordinated Co(R) compound. In contrast to the free cobalt tetrasulfonated phthalocyanine, its complex with apocytochrome c has not the ability to reverse oxygen binding. Reduced with dithionite or ascorbate, however, it combines with oxygen-giving oxygen adduct. That suggests that the modified cobalt phtbaiocyanine cytochrome c in its reduced form possesses a structuraifeature in common with the oxygencarrying heme proteins. It also resembIes that of cytocbrome c with alkylated methionines 127, 281, where the sulfur of methionine-80 is displaced from the crevice-closing position. Reduced iron phthalocyanine complex in aerobic conditions is oxidized directly to the
ferric state. Incorporation
of
the
iron
and
cobalt
tetrasulfonated
phthalocyanines
into
apocytochrome c changes unfo!ded conformation of the protein toward a more ordered structure_It is, however, significantly alteredas compared with the native cytochromec. The calculation of the helical contents from the present CD spectra in acetate buffer, compared with that of cytochrome c taken at about 28.6% [IO], gives values for the percent -helical structurein iron and cobalt phthalocyaninemodels of about 9.23 and 10, respectively. In 50% 2chlorethanol, helicity of these complexes is also Iower than that of the native cytochrome c. Stabilizationof the apocytochromec structureowing to its coordination with phthalocyaninederivativesis demonstratedby the bigher resistance of the model complexes to denaturation as compated with apocytochrome c. The electrophoretic mobilities of the model complexes are nearly identical with that of the native enzyme indicating ah these species to have the same charge at the surface of the protein. Molecular weights of the examined complexes are close to thatof cytochromec suggesting thatthey are monomers_ The midpoint potentials for the iron and cobalt phtbaioeyaninemodels were found to be -f-374 and +320 mV, respectively. Contrary to thatbetween the native cytochromec and its cobalt substitutedanalog, the difference m the redox potentials between both examined complexes is relatively small and is close to thatbetween hemoglobin (E,, = f 147 mV) and coboglobin (Em_,= + 100 mV). Beef mitochondriai cytochrome c reductase reduces both complexes immediately after mixing the reactants.They are reduced also by cytochrome c(E,,,~ = +260 mV). More detailed investigations of rhe enzymic activity of the model complexes will be reported in the following papers. The autooxidizability of the ferrous phthakXyaninemode1and rapid formation of the oxygen complex by its
I. B. M. Hoffmanand D. H. Petering,??oc IVatL Acad Sci USA 67,637 ;1970)_
Tetrasulfonated
2. 3.
Phthalocyanines
5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28.
c
199
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S_ F_ Andres and M_ 2. Atassi. Biochemistry 9.2268 (1970). J. L. Leonard, T. Yonetani, and J. B. Callis, Biochemistry 13, 1460 (1974). L. C. Dickinson and J. C. W. Cbien, Biochem. Biophyx Res. Commun. 58,235 (1974). J. C. W. Chien, L. C. Dickinson, and T. L. Msson, Biochem. Biophys. Res. Commun_ 63,853
(1974). L. C. Dickinson and J. C. W. Chien, Biochemistry 14.3526 (1975). L. C. Dickinson and J. C. W. Cbien. Biochemistry 14.3534 (1975). M. C. Findlay and J. C. W. Cbien. Eur. J_ Biochem. 76,79 (1977). J. M. Vanderkooi, F. Adar, and M. Erecinska, Ew_ J. Biochem. 64,381 (1976). J. C. W. Chien, l-L L. Gibson, and L. C. Dickinson, Biochemistry 17,2579 (1978). A_ M_ Scbaffer, M. Gouterman, and E_ R_ Davidson, Theor. Chim. Acfa 30.9 (1973). H. Prrywarska-Boniecka, L. Trynda, and E. Antonini, Eur. J. Biochem 52,567 (1975). H. Przywarska-Boniecka and L. Trynda, Eur. J. Biochem. 87,569 (1978). H. F’rzywarskc-Boniecka and L. Trynda (in press). W. R. Fisher, H. Taniuchi, and C. B. Anfiin, J. Biol. Chem. 248,3188 (1973). E. SteBwagen, R. Rysavy, and G. Babul. J_ Biol. Chem_ 247,8074 (1972). T. Yonetani,J. BioL Chem. 235,845 (1960). C. A. Yu and T. E. King, J. BioI_ Chem. 243,490s (1974). D. Vonderschmitt, K. Bemauer, and S. FaUab, Helv. Chim Acta 48, 951 (1965). E. Anton!, J. Wyman, M. Brunori, J. F. Taylor, A_ Rossi-Fanelli, and A_ Caputo, J. Biol. Chem. 239,907 (1964). N. M. Clark, Oxidation-Reduction Potentials of Organic Systems. Williams and Wilkins, Baltimore, 1960, p_ 45.5. D. W. Clark and J. R. Yandle, Znorg. Chem. 11,1738 (1972). H. Przywarska-Boniecka, E. Panejko, L. Trynda, and J. Biemat,Mater_ Sci. 4, 139 (1978). C_ Tonzolo, A_ Fontara and E Scoffone, Eur. J_ Biochem 50,367 (1975). A_ Scbejter and I. Aviram. J. BioL Chern. 245,1552 (1970). M. T. Wilson, M. Brunori, G. C_ Rotilio, and E. Antonini, J. BioL Chem. 248,8162 (1973).
ABSTRACT ArtificiaI cytochromes c have been prepared with Fe(III) and Co(ilI) tetrasulfonated phthalocyanines in place of heme. Their structure and properties have been lnvestigated by difference spectroscopy, CD, epr, electrophoresis, molecuIar weight estimation, and potentiometric measzements. The visiile absorption spectra show the main peak at 650 mn for the iron compound 685 nm for the cobalt one. It is shown by CD experiments that incorporation of Fe(III)L or Co(III)L into apocytochrome c markedly increases helical content of the protein. Its conformation is, however, significantly altered as compared with the native cytochrome c. The epr and spectroscopic data show that the iron and cobalt phthalocyanine models represent the low spin species with the metal ions in trivalent state. Eiectrophoresis and molecular weight estimation indicate these complexes to be monomers_ Both phthalocyanine complexes have not affinity for additional ligands characteristic for hemoglobin_ They react, however, with CO, NO, and CN- whin they are reduced with dithionite. Moreover. Co(II)L-apocyt c is able to combine with oxygen suggesting a structural feature in common with the oxygen-carrying heme proteins. Iron complex in the same conditions is oxidized directly to the ferric state. The half-reduction potentials of Fe(III)L-apocyt c and Co(III)L-apocyt c are +374 mV and +320 mV, respectively. These complexes are reduced by cytochrome c and cytochrome c reductase (cytochrome bq).
Abbreviations L, tetranrlfonated phthalocyanine ligand [C32H12N&03Na)4] ; Fe(III)L, iron(M) tetrasuIfonated phthaiocyaniue; Co(III)L, cobalt(III) tetrasulfonated phthalocyanine; apocyt c, apocytochrome c.
INTRODUCTION Specific modifi~tions of the native metalioenzymes have been accepted as one of the most usefui methods to study function-structux relationships of these compounds. Address reprint requests to Docdr hab_ Helena Boniecka, htytut ul.F.JoIiotCurie 14, SO-383 Wroolaw_ Jo-~of~norg~ic~~~~~m~~16,183-199 0 Elsevier Science Pub!ishiug Cp., Inc_, 1982
52 VandzrbiIt Ave., yew York, NY 10017
Chemii Uniwersytetu Wrodawskiego,
(1981)
183 0162-0134/82/030183-1752.75
184
H. Przywarska-Boniecka and L. Ostropolska
Recent studies have shown thatit is rassible to modify hemoglobins. myoglobins, [l-5], and cytochrorue c [cl I] by inco.porating the protoporphyrin of metal other than iron into the heme crevice of their apoproteiris. Investigations of these metal substituted derivatives gave valuable infomations about mechanism of the allosteric transition in hemoglobin and shed light on the requirements for electron transfer processes in CJtochrome systems [ 123. Cobalt derivatives allowed epr studies which are not possible fclr diamagnetic ferrous species. The compounds which closely resemble iron porphyrin are iron and cobalt pi~thaic~ya-ines as well as their water soluble tetrasulfonatedderivatives [ 131. Earlier we have shown that substitution of heme in hemoglobin by metal tetrasulfonated phthaiocvanines gives dimeric complexes v/hose properties resemble those of the native species [14, 151. Combination of iron phtha!ocyanine derivative with apocatalase, however, gives denatured species [ 163_ We now report the re~lts concerning the preparation, structure, and properties of artificial cytocbromes obtained by incorporation of iron and cobalt tetrasulfonated phthalocyanines into the heme binding site of a-3 tochrome c _
Pig heart cytxhrome cbromato@fic~y
c was purchad
from Biomed (Krakow) and purified
[ 17 J.
Apocyt~~trome c was prepared by modification OFthe silver sulfate method [IS]specifically, 160 mg of Ag$O, in IS ml of water am! 1 6 ml of acetic acid were added to 100 mg of cytochrome c disolved in 2 ml of water. The solution was incubated in the dark for 4 hr a& 44°C zd was then centritiged to remove heme aggregates. The apoc:ytocLmme t was separated from the heme and precipitatedby addition of 15 ml of cold
Tetrasulfonated Phthalocyanines and -4pocytochrome c
185
The synthesis of the metal phthalocyanine substituted cytochmme c involves the incubation of apocytochrome c with twice excess of the metal tetrasulfonated phthalocyanine in 0.2 M acetic acid for 5 days at 4°C. The reaction mixture was separated by gel filtration on Sephadex G-50 and the fracticns were identified by absorption spectroscopy at 280 and 650 or 685 run, respectively, for the iron and cobalt derivatives. The bluegreen protein fraction were iyophilized. The reduced forms of the complexes were prepared by addition of a few milligrams of sodium dithionite to the solutions and removing the excess of the reductanton a Sephadex G-50 column in argon atmosphere. The concentrations of the complexes were determined from the molar absorption coefficients. To avoid errors in concentrations due to the adsorption of the free-metal phtbalocyanine impurityon the protein surface, molar absorption coefficients were determined for every portion of preparation. Their average values were thus: Fe(III)L-apocyt c, em = 2.6 x 101 M-l cm-‘; Co(JII)L-apocyt c, esss3.75 x 101M-r cm-*, Fe(II)L-apocyt c, ~~ = 3.16 x l(r M-* cm-r; Co(II)L-apocyt c, r,, = 2.9 x l(r M-r cm-r _ Hemin was obtained from Koch-Light Chemical Co. The combination of the metal phthakyanine substitutedcqtochromes c with nitric oxide was carried out anaerobically. Purified argon was passed through the solution for 20 mitt and then was replaced with nitric oxide by passing NO through the solution for a further 20 min. Evaporated water was completed in anaerobic conditions at 4°C. The sample was allowed to stand for 24 hr before use in spectroscopic and epr experiments.
Absorption
Spectroscopy
Absorption and difference spectra were performed on a Car-r 15 recording spectrometer with a cell compartment thermostatically controiled at 15°C or on Specord recording spectrophotometer. The measurementswere made under equilibnzm conditions. Reaction with Hemin The reaction of Fe(IJJ)L-apocyt c with hemin was carried out in molar radioof complex to hemin 1:4. The reaction mixture was incubated at 4”1=in the dark for 14 days and then separated chromatographically. It was applied to a column of Sephadex G-50 equilibrated with O-05 M acetate buffer pH 5.8. Elution was carried out with the same buffer at a flow rate of 25 ml/hr. Three-milliliter fractions were collected and examined spectrophotometrically. The gel filtration profile was made for A,, A,,, , and A,, _ Cinrillar Dichroism Measurements Circular dichroism (CD) spectra were recorded using a model ORD/uv-5 Japan Spectropolatimeter with CD attachment. The solutions were prepared by dissclving appropriate amounts of lyophilizated preparation in acetate buffer, pH 5.8, or in 50% cbloroetllanol. EIktl7lllP :aramagnetic Resonance (epr) X-band epr spectra of the complexes in buffered solutions were performed at liquid nitrogen temperaturewith Varian model JES-ME-3X spectrometer.The solutions under
186
H. Przywarska-Boniecka and L. Ostropolska
investigation in these conditions become fi-czen and, experimentally, they behave as glassy media. The microwave frequency was 9.27 GHz. The epr spectra of the reduced complexes or their NO derivatives were made in anaerobic conditions. PoIy-crylamide Gel F.&ctrophoresis Electrophoretic studies were carried out according to the method described earlier [ 151.
Moh=cdar Weight Estimation Molecular weights of the model compleres wete determined by gel filtration on Sephadex G-75 column according to the method of Andrews (see Stellwagen et al [ 181). I he following proteins were used as reference substances: insulin (MW 6,000), cytochrome c (Mw 12,400), lyzozyme (MW 14,000), myoglobin (MW 17,8CK!),pepsin (MW 35,000), and serum albumin (MW 67,000).
OxidatiorkReduction
Potentials
The oxidation+eduction potentials of the model complexes were studied by mixture method [22] which consists of the measurements of the potential of mixtures of the oxmixed and reduced forms of the examined compound in known proportion. The fully reduced preparations were obtained according to the method described earlier !8]_ The full oxidation was performed by addition of stoichiometric amounfj of K3 [Fe(CN),] to tie soiutions of the examined complexes [5 x 10-j M] and separation of the oxidation pro&co on a Sephadex G-50 coiumn equilibrated with 0.005 M phosphate buffer, pH 7, and 0.2 M KC1 _ The molar concentrations of the complexes were determiner; from the molar absorption coefficients. Oxidatio-reduction pc:entials were determmed in a titration chamber similar to those employed by Clark [23]. it was fitted for argon purge, platinum flag electrode, and saturated calo.mel electrode with a KCl-agar salt bridge. Voltages were measured with type V-627 potentiometer. Absorbance was followed by Cary 15 spectrometer. A mixture of mediators was used f0.5 PM] to facilitate the equilibration of the oxidize&reduced forms. They are Ks[Fe(CNX], methylene blue, thionine, and to!uene blue. A known amount of fully oxidized complex in buffered solution was titrated in argon atmosphere with increasing amounts of its reduced form,
Assay
Enzymatic
Enzymic reduction of Fe(III)L-apocyt c and Co(iII)L-apocyt c was assayed with system involving mitochondrial cytocbrome 5 reductase and NADH as a substrate, disso,ved in 0.1 M phosphate buffer, pH 7, made 0.1% in Triton X-100. All solutions were deoxygenateti by argon purging over 2 hr. The assays were performed by injection anaerobically appropriate quantiries of NADH and cytocbrome c reductase (bc, = [2.2 x lo-“, MJ, complexess = 11.2 x IO- 5, Ml). The reaction run was followed spect.rophGtometrically.
RESULTS Artificial cytochromes c cannot be obtained by a displacement of heme in cytochrome c with metal tetrasulfonated phthalocyanines. Instead, phthalocyanine derivatives are
Tetrasulfonated
Phthalocyanines
and Apocytochrome
c
187
reduced with cytochrome c as is observed in the difference spectra presented in Figure 1. Characteristic absorption peak for iron tetrasulfonated phthalocyanine in water solution is at 635 run. Water solutions of cobaIt(II) tetrasulfonated phthalocyanine contain an equilibrium of monomer and dimer with characteristic absorptions at 668 and 625 run. These peaks disappear when cytochrome c is added to such solutions and new ones arise at the same time, at 670 nm for the iron complex and at 698 and 740 nm for the cobalt one. The position of the new bands points to the reduction of the phthalocyanine complexes 124,251. Cytocbrome c, on the other hand, is oxidized to its ferric form as is demonstrated by disappearance of its band at 424 nm and the arise of another one at 408 mn which is characteristic for the ferric cymchrome c. I’nteraction of Iron and Cobalt Phtbalocyanine Complexes (Fe(IIZ)L and
Co(Il)L) with Apocytochrome
c
The reaction between metal tetrasuifonated
phthaiocyanines and apocytochrome c was proved by the difference spectroscopy experimenrs. Visible difference spectrum of Fe(IIi)L-apocytochrome c system shows two positive peaks at 650 and 695 nm, and a negative one at 635 mn. This points to the formation of the complex between phthalocyanine derivative and apocyt~hrome c (Fig. 2). Visible difference spechurn of Co(II)L-apocytochrome c system exhibits a positive peak at 685 nm and two negative peaks at 625 and (68 nm. Spectrophotometric titration data indicate the formation of the metal-phthalocyanine-apocytmk-ome c cor;pIexes at the mokir ratio of the reactants 111. Incubation of Fe(III)L-apocyt c with hemin in acetate buffer pH 5.8 for 2 weeks leads to the displacement of Fe(III)L by hemin and reconstitution of cytochrome c. The results of the chromatografic separation of the reaction mixture are presented in Figure 3. The eiution profile exhibits three well-resolved peaks at the positions of 72(I), 84(II), and 162 ml(III). Two leading fractions are proteins with charac+tic absorptions at 650(I) and 413 nm(II). Fraction I which appears at the solvent front is blue and has been identified as Fe(III)I-apocyt c. Fraction II exhibits abmrption spectrum corresponding to that of cytochmme c. The lower moIecular weight peak represents the bIue fraction (III) with characteristic absorption, a:. 635 nm. It has been identified as Fe(III)L. The unreacted hemin remains on the coiumn graduaiIy spreading from the origin. The affinity of the model complexes for additional ligands were studied spectroscopically. In experimental conditions, they do not have the ability to coordinate with imidazole, CO, NO, and CN- _ They react, however, with these iigands when they are reduced, this is demonsL;ated by the increase of intensity of their main absorption bands with minimal batochromic shift. The complex of cobalt tetrasulfonated phthakyanine with apocytochrome c virtually has not the ability to reverse the oxygen binding, suggesting that an insertion of Co(II)L into apoprotein in aerobic conditions leads to oxidation of cobalt ion to its trivalent state. The phtbalocyanine substituted cytochromes c are reduced with dithionite as is shown by characteristic changes in their absorption spectra iFis_ 4). Figure 4A presents visible difference specaum of Fe(III)capocyt c reduced with dithionite against the same complex -unreduced. The reduced form exhibits two absorption bands at 680 and 608 NTL This skxtrurn rrrzverts into that of Fe(III)L-apocyt c in an air atmosphere.
The reductton of Co(III)L-apocyt c with dithionite in argon results in a disappearance of thy band at 685 nm and an arise of a new one at 638 nm (Fig. 4B). On exposure to
) mixturesagainstthe snmeaolutlons FIGURE 1. Differencespectra of Fe(lll)L t ferrous cytochrome c (-- ) nnd Co(ll)L t ferrous cytochrome c 6-v unmixed.R(III)L = Co(II)L = (8.6 x 10-s Mj, ferrous cytochromec = [5,54 X 10-e M].
Ak
Tetrasuifonated Phthalocyaninas and Apocytochrome
c
189
(nm 1
FIGURE 2. Difference spectra of ke(III)L + apocytochrome c (-) and Co(II)L + apocytochrome mixtures against the same solutions unmixed: Fe(III)L = Co(II)L = [2.0 x 104 M] , apoc (---) cytochromec= [LOX 10-5&f].
oxygen, reconstitution of the band at 685 nm is observed. It is, however, more intense than that of the original complex. The band at 638 nm disappears at the same time. When bubbled with argon, the oxygenated solution zailses a gradual decrease of the absorption at 685 run. Prolonged bubbling with oxygen leads to an irreversible oxidation of the cobalt ion. The spectrophotometric experiments indicate that Co(III)L-apocyt c reduced with dithionite can combine reversibly with oxygen, giving an oxygenated intermediate_ Similar results are obtained with an ascorbate as the reductant, but in this case the reaction is slower than that with dithionite.
!Sctmphoresis
and Molemhr
Weight Estimation
Polyacrylamide gel electrophoresis patterns show that the electrophoretic mobilities of both model complexes are identical with that of the native enzyme. The results of the molecular weight estimation of Fe(III)L-apoc: t c and Co(III)Lapocyt c show that the molecular weight of the iron complex is approximately 13,000 and that of the cobalt complex is 12,!JOO.These values are comparable with that of cytochrome c.
circuIarDichroismspectM The conformationaL properties of the model complexes were examined by CD technique in acetate buffer, pH 5.8. and in 50% 2chloroethanol. For comparative purposes, CD spectra of apocytochrome c and native cytochrome c were performed at the same
FIGURE 3. The gel fdtrction profile of the reaction products of F’c(lll)L-apocytc with hemin in 0,05 M acetate buffer, pH 5.8, on a column (2 x 85) of ScphadexG-50at 25 ml/hr, 4°C I,&~o; II,,4413; ill,A6g6,
Frraction number 9
6. r
Tetrasulfonated Phthalocyanines and Apocytochrome c ,
191
FIGIiRE 4. Differencespectraof (A) Fe(IIDL-apocytc + ??a&$4
and (B) Co(III)L-apocyt c + Na2S204 mixtures against the same solutions of the complexes without dithionite, in argon (-) and in aerobic conditions (---)_ Fe(iJI)L-apocyt c = 12.4 x 10-5 Ml, Co
conditior;s. In acetatebuffer, apocytochrome c exhibits a large negative maximum near 200 run accompanied by negative infkction near 225 MI. This patternis indicative of a predominantly unordered conformation. In 2-chloroethanoI. however, CD spectrumof apocytochrome c is characterizd by two intense negative bands at about 210 and 223 nm. Both spectra are in agreement with those previously reported 1261. Native cytocbrome c displays a strong Cotton effects at 210 and 223 nm in acetate buffer as welI as in 2-chloroethanol_ Ellipticity vaiues expressed in deg cm* dmol-’ are -7.9 x 105 in acetate buffer (Fig. 5A), Btlo = - 1.85 X 106, 8t,0=-6~105,8tu= 9 tu = -2.25 X 106in 2-chlomethanole (Fig. 5b& The phthalocyaninemodel complexes exhibit negative uv Cotton effects at 210 and 223 nm in both solvents. The elliptic@ values, however, are significantly lower than those of cytochmme c. In acetate buffer they are & = -3.0 x 105,em = -2.7 x 105for the cobahic complex, & = -3.4 X
192
H. Przywarska-Boniecka
and L. Ostropolska
-
300 WWeiE!!gh
x)
210
220
230
W avelengh
240
(nm)
-@
(nm) -240
Fe(III)LFIGURE 5. Ultraviolet CD spectra of cytochrome c (-- ), apocytochmme c (--), apocyt c (------). and Co(III)L-apocyt c (-----;) in (A) 0.2 M acetate buffer, pH 5.8, and (J3)in 2-chhoethanok. (Cl CD spectraof the model compiexesin acetatebuffer io the Soret region
Tetrasulfcmated Phthdlocyznines and Apocytochrome
c
193
em=
-3.2 X f(Y for the ferric one. The elliptic@ values for the ferric compIex in 105, 50% 2chloroethanol are &,,, = - 1.65 x 106, t& = 1.85 x 106. All ellipticity values are expressed in deg cm’ dmol-1 . In the Soret regian (300420 nm) the model complexes in acetate buffer both display induced Cotton effects with two positive CD bands of identical intensity (Fig. 5C). They are observed at 315 and 333 nm in CD spectrum of Co(iII)L-apocytochrome c (0 = 2.1 x 104 deg cm’ dmol -1) and at 335 and 347 run in that of Fe(III)Capocyt c (e = 2.1 x 1oQdeg cm* dmol -I)_ The absorption spectra of these complexes show in this region one broad band which appears at 325 nm for the cobalt complex and at 342 nm for the iron one. Electron ParamagneticResonance Study The epr studies of Fe(lIT)L-apocyt c at pH 5.8, 77”K, shows distinct broadened low spin signal with a central g value of 2.106 (Fig. 6). It has no observable hypetfine splitting- Asymmetric shape of this signal could suggest superpositionof the signals of two low spin species. Afterzduction with dithionite, epr spectrum disappears due to low spin Fe(iI)L-apocyt c. Nitric oxide derivative of Fe(II)L-apocyt c exhibits an intense epr spectrum with the central resonance signal at g = 2.084. The splitting from the hypctfine interaction with nitrogen nucleus of the NO Iigand is not observed in this spectrum indicating that the unpaired electron of NO spends a considerable fractionof time in the iron orbitals. The epr spectrum of the cobalt phthalocyanine model of cytochrome c at pH 5.8 is more complicated (Fig. 7A). It consists of two broad, weak signals at the low and high field regions. The weaker low field signal exhibits g = 3.53. The high field signal is more intense and shows partially resolved hyperfine structure. Its spin Hamiltonian parametersareg = 2.31,g = 2.03, A = 39G, A = 91 G. Additionofdithionite to the examined solution produces the characteristic change of its epr spectrum (Fig. 7B). The intensity of the low spin signal markedly increases what points to the reduction of the majority of the original preparation. Beside the hyperfine structure partially resolved, supe .hyperfiie lines from one nitrogen of the axial ligand are observedThe epr study results suggest that the main species of the preparation constitutes Co(III) complex. The low field minority component of epr spectrum is probably due to impurity of Co(II)L coordinated to some unspecific protein residue.
oxidation-Reduction
Potentials
Figure 8 shows oxidatiorr-reduction equilibrium curves of the complexes of iron and cobalt tetrasulfonated phthalocyanines with apocytochrome c at 25°C as obtained by the mixture method. Methylene blue was used as mediator in these experiments. The values of &Xl., given by the oxidizing titrations are +375 and t320 mV for iron and cobalt complexes, respectively. I&se values are reproduced in the reducing titrations and in the presence of the mixture of the following mediators: K,Fe
H. Przywarska-Boniecka
194
and L. Ostropolska
>
3m FIGURE 6. X-band epr spectn of (A) Fe(iII)L-apocyt buffer, pH 5.8, at liquid nitrogen temperature.
__3mo
3300
H
c and Q) its NO derivative in 0.2 M acette
reaction mixtures (Fig. 9). They show a rapid decree of the band characteristicfor the oxidized form of the model complexes and an appearanceof thatcorresponding to their reduced form. Simuhaxously, growth of the band at 424 MI characteristicfor the oxidized cytochrome reductase is observed. The e xamhed complexes are reduced ako Sy the native qtochrome c; this is in agreement with their redox potentials (A?& of cytochrome c is t260 mV).
Tetrasulfonated
PhthalocyaGnes
and Apocytochrome
c
195
FIGURE 7. X-band epr spectraof Co(III)L-apocyt tin 0.2 M acetate buffer, pH 5.8, (Al in original form, (Is) rexiucedwith dithionite.
H. Przywarska-Boniecka and L. Ostropolska
196
FIGURE 8_Oxidatio=-reduction titration of Fe(lII)L-apocyt c ( -) and Co
CONCLU§ION The interaction between the iron and cobalt tetrasulfonated phthalocyanines and apocytochrome c leads to the formation of artificial cytochromes c with the iron and robalr phtbalocyanine derivatives in place of protoheme. The visible spectra of these complexes are characteriz& by a main intensepeak at 650 mn for the ferric compound and at 685 nm for the cobalt one. In addition, the ferric complex exhibits a weaker band at 695 nm. Tbe 695 nm band in the ferric cytoehrome c is considered to he strong evidence for the ligr tion of methicnine-80. However, it is relatively intensein Fe(III)Lapoeyt c and cannot be assigned to the Fe-S bond without additional proofs. In contrary to hemoglobin, heme in cytochrome c cannot be displaced by the metal phtbalocyanines. It is covalently bonded to the protein by strong thioether bridges between the porphyrin ring and two cysteine residues in the peptide chain. Phthaloeyanines do not have the capability to form such bonds. Instead, metal tetrasulfonatedphthalocyanines are removed from the model complexes by hemin with reconstitution of the ferric cytochrome c. This fact suggests that the metal phtbalocyanine binding site in apocytochrome c is the same as that of hemin in cytochrome cc _ The identification of the axial ligands in the examined complexes is uncertain at present. They are not displaced by such molecules as CO, NO, cyanide, and imidazole; they then seem to he strong field ligands such as histidine, lysine, or me&ion&. In the reduced form, however, both phthalocyaninecomplexes can readily bind additional Iigands. It is assumed thatthe reactionof the examined complexes with ditbionite or ascotbate causes conformational change that leads to the loosening of the
Tetrasulfonated
Phthalocyaninx
and Apocytochrome
axJoq1osq\d
197
c
0
H. Przywarska-Boniecka and L. Ostropolska
198
heme crevice relative to the original species_As a result, a substitutionof an axial ligand by another from outside is easily made. The iron and cobalt phthalocyanine models both represent the low spin species. The epr spectra indicate that the cobalt ion in the model complex appears mainly in the trivalent state and reduction with dithionite yields a Co(R) state. Some portion of the original preparationexists as a six-coordinated Co(R) compound. In contrast to the free cobalt tetrasulfonated phthalocyanine, its complex with apocytochrome c has not the ability to reverse oxygen binding. Reduced with dithionite or ascorbate, however, it combines with oxygen-giving oxygen adduct. That suggests that the modified cobalt phtbaiocyanine cytochrome c in its reduced form possesses a structuraifeature in common with the oxygencarrying heme proteins. It also resembIes that of cytocbrome c with alkylated methionines 127, 281, where the sulfur of methionine-80 is displaced from the crevice-closing position. Reduced iron phthalocyanine complex in aerobic conditions is oxidized directly to the
ferric state. Incorporation
of
the
iron
and
cobalt
tetrasulfonated
phthalocyanines
into
apocytochrome c changes unfo!ded conformation of the protein toward a more ordered structure_It is, however, significantly alteredas compared with the native cytochromec. The calculation of the helical contents from the present CD spectra in acetate buffer, compared with that of cytochrome c taken at about 28.6% [IO], gives values for the percent -helical structurein iron and cobalt phthalocyaninemodels of about 9.23 and 10, respectively. In 50% 2chlorethanol, helicity of these complexes is also Iower than that of the native cytochrome c. Stabilizationof the apocytochromec structureowing to its coordination with phthalocyaninederivativesis demonstratedby the bigher resistance of the model complexes to denaturation as compated with apocytochrome c. The electrophoretic mobilities of the model complexes are nearly identical with that of the native enzyme indicating ah these species to have the same charge at the surface of the protein. Molecular weights of the examined complexes are close to thatof cytochromec suggesting thatthey are monomers_ The midpoint potentials for the iron and cobalt phtbaioeyaninemodels were found to be -f-374 and +320 mV, respectively. Contrary to thatbetween the native cytochromec and its cobalt substitutedanalog, the difference m the redox potentials between both examined complexes is relatively small and is close to thatbetween hemoglobin (E,, = f 147 mV) and coboglobin (Em_,= + 100 mV). Beef mitochondriai cytochrome c reductase reduces both complexes immediately after mixing the reactants.They are reduced also by cytochrome c(E,,,~ = +260 mV). More detailed investigations of rhe enzymic activity of the model complexes will be reported in the following papers. The autooxidizability of the ferrous phthakXyaninemode1and rapid formation of the oxygen complex by its
I. B. M. Hoffmanand D. H. Petering,??oc IVatL Acad Sci USA 67,637 ;1970)_
Tetrasulfonated
2. 3.
Phthalocyanines
5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28.
c
199
T. L. Fabry, C. Simo, and K. Javaherian, Biochim. Biophys Acfa 160.118 (1968). T_ Yonetani, H. R. Drott, J. S. Leigh, G. Reed, M. R. Waterman, and T. Asacura, L Biol.
Chem. 245,2998 4.
and Apocytochrome
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