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Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode
J ~ R N A L OF
ELSEVIER
Journal of Electroanalytical Chemistry 420 (1997) 5-9
Preliminary note
Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode Isao Taniguchi a,*, Yasuhiro Mie a, Katsuhiko Nishiyama a, Viktor Brabec a,l, Olga Novakova 1 a, Saburo Neya b, Noriaki Funasaki b a Department of Applied Chemistry and Biochemistry. Faculty of Engineering, Kumamoto University, Kurokami, Kumamoto 860, Japan b Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan Received 7 August 1996; revised 30 September 1996
Abstract
Monoazahemin reconstituted myoglobin was prepared and its electrochemical behavior was studied in comparison with native myoglobin. For both myoglobins well-defined voltammograms were clearly obtained at highly hydrophilic surfaces of indium oxide electrodes. Although monoazahemin showed a more positive redox potential than hemin (measured in methanol), monoazahemin reconstituted myoglobin showed a more negative redox potential than native myoglobin in a 50 mM bis-Tris buffer solution (pH 6.5), suggesting that for both native and reconstituted myoglobins the heme environment including proximal histidine as an axial ligand of the redox center plays an important role in determining the redox potential. Also, a unique electrochemical response of cyano-monoazahemin reconstituted myoglobin was demonstrated. Keywords: Myoglobin; Monoazahemin; Reconstituted myoglobin; Indium oxide; Redox potential; Cyclic voltammetry
1. Introduction Recently, electron transfer reactions of metalioproteins at functional electrodes have become one of the most attractive fields in electrochemistry and related fields of chemistry (see for example Ref. [1]), and various electrode reactions of electron transfer proteins have been reported [2-8]. However, electrochemical studies of metalloproteins with biological functions other than electron transfer have still been very limited. Myoglobin (Mb), for example, is a medium-sized heme protein having biological functions of dioxygen storage and transport, and its rapid direct electron transfer at an electrode has been unsuccessful until fairly recently. We have found that Mb from both horse heart and sperm whale skeletal muscle gave well-defined quasi-reversible voltammograms at the highly hydrophilic surface of an In203 electrode [9,10]. Mb is a very attractive biomolecule, not only because of its native physiological functions but also because of the
possible introduction of new artificial functions. The redox center of Mb is located at the heme pocket with no covalent binding to the globin moiety, and therefore can easily be replaced by other molecules to prepare semiartificial myoglobins. In the present study Mb, of which the redox center (hemin) was substituted with the iron complex of ctazamesoporphyrin XIII (monoazahemin), i.e. monoazahemin reconstituted myoglobin (azaMb) [11], has been prepared, and its unique redox responses were observed for the first time in comparison with native metMb. For native and reconstituted myoglobins, the axial ligands of the redox center as well as the porphyrin ring are clearly demonstrated to be important for their electrochemical reactions. For example, although monoazahemin itself has a more positive redox potential than hemin, azaMb showed a more negative redox potential than native Mb.
2. Experimental " Corresponding author. On leave from the Institute of Biophysics, Academy of Sciences, Brno (Czech Republic) as Visiting Research Scholars of the HeiwaNakashima Foundation (VB) and JSPS Research Fellow (ON).
Horse heart Mb was obtained from Sigma, and was purified further by chromatography [12,13] on a Whatman CM-52 column at 4°C using a phosphate buffer solution
0022-0728/97/$17.00 Copyright © 1997 Elsevier Science S.A. All rights reserved. PH S 0 0 2 2 - 0 7 2 8 ( 9 6 ) 0 1 0 2 2 - 4
6
1. Taniguchi et al./Journal of Electroanalytical Chemistry 420 (1997) 5 - 9
(16mM N a H z P O 4 + 3 . 1 m M N%HPO4, pH 6.5; ionic strength /z = ca. 0.025 M) as eluent. The Mb obtained was dialyzed against distilled water and then concentrated by ultrafiltration. The SDS-PAGE measurement of Mb thus obtained gave a single band having an appropriate molecular weight (ca. 17000). Various spectrochemical data showed the sample was native and pure. The concentrations of myoglobin were estimated spectroscopically for ferro Mb (reduced with sodium dithionite) at pH 6.5 using the molar absorptivity of l l 4 0 0 0 M - ~ c m -1 at 434nm [12,13]. Apomyoglobin (apoMb) was prepared from horse heart Mb by a similar procedure to that described in the literature [11] on the basis of the acid methylethylketone method [14,15], and dialyzed against an excess amount of 10mM cold bis-Tris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol) buffer solution (pH 6.5) for more than 24h to remove methylethyiketone. Monoazahemin (see Fig. 1 for its structure) was synthesized according to the literature [l 1]. ApoMb obtained was then mixed in an ice bath with a 1.2 molar equivalent amount of the monoazahemin dissolved in methanol, and dialyzed with an excess amount of the bis-Tris buffer solution overnight to obtain azaMb. AzaMb was then purified by co]umn chromatography on a Whatman CM-52 column below 4°C. The spectrum of the azaMb (Fig. l) was the same as that reported previously [11], and the purity was satisfactory [absorbance ratio A(Soret = 385 nm)/A(280nm) = 3.5, which is smaller than that (5.0) of native Mb]. In the present study, especially for electrochemical use, only fractions having absorbance ratio 3.5 were used. All other reagents used were of analytical grade. 25 to 50mM bis-Tris buffer solutions were used for electro-
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600
Wavelength / nm
Fig. I. UV-visible spectra of purified (---) native myoglobin, ( ) monoazahemin reconstituted myoglobin (in ferric forms) and ( - - - ) apomyoglobinin a 50raM bis-Tris buffer solution (pH 6.5), together with the structures of heroin and monoazahemin as prosthetic groups.
chemical measurements, where the electrochemical responses were independent of the buffer concentrations. Cyclic voitammograms were obtained at 25°C using a BAS CV-50W electrochemical system at an In20 3 electrode (approximately 5 X 5 m m 2, from Kinoene Optics Corp., Japan) under nitrogen atmosphere. The electrode was cleaned by ultrasonication in a 1% aqueous New-Vista (anionic surfactant, AIC Corp.) solution, followed by ultrasonic washing in distilled water until the surface of the electrode became fully hydrophilic. The surface hydrophilicity was monitored by measuring the surface tension of the electrode by the Wilhelmy method using a Shimadzu ST-I surface tensometer [10] to be more than 7 0 d y n c m -t at 25°C in water (cf. the surface tension of water itself at 25°C, equal to 72 dyn c m - a). A Pt plate and an AglAgCIIKC1sat were used as the counter and reference electrodes respectively. The UV-visible and circular dichroism (CD) spectra were measured using Shimadzu UV-2100 and JASCO J-720 spectrophotometers respectively.
3. Results and discussion
For metMb electrochemistry, the surface hydrophilicity of an In203 electrode affected very much the peak separation of the voltammogram, and almost fully hydrophilic surfaces ( > 70 dyn c m - ~) of In203 electrodes were useful in giving well-defined voltammograms for both sperm whale skeletal muscle and horse heart metMb with a heterogeneous electron transfer rate constant k ~' of ca. (1 to 3 ) × 10 -4 c m s - ~ at pH 6.5, as reported previously [9,10]. Similarly, for azaMb, well-defined voltammograms were obtained at a highly hydrophilic surface of In203 in a 50 mM bis-Tris buffer solution (pH 6.5) as shown in Fig. 2(a). No significant change in the voltammograms was observed during the continuous measurements for at least an hour. The peak current was linear with the concentration of the azaMb tested (up to ca. 0.1 mM), and was proportional to the square root of the scan rate, u ~/2, in the u region up to 100mVs-~. At 25°C the diffusion coefficient D of azaMb, estimated by both cyclic voltammetry and potential-step chronocoulometry after correction of the active area of the electrode surface from the geometric one, was ca. 9.0( _ 1.0) × 10- 7 cm 2 s- l, which is in good agreement with that reported for sperm whale Mb (1.1 × 10 -6 cm 2 s-1 [16]). The formal redox potential E ~ was evaluated, as the midpoint of anodic and cathodic peak potentials of the redox wave, to be - 0 . 1 8 V vs. Ag [AgCIIKCIsa t, which is more negative than that of native Mb by ca. 40mV. This point will be discussed in more detail later in the paper. The observed voltammogram showed that the reaction was quasi-reversible, and the k~' value of ca. 2 x 10 4 cm s- ~ was estimated, using a digital simulation technique (see Fig. 2(b)), to be similar to that of native Mb [9,10].
I. Taniguchi et al./ Journal of Electroanalytical Chemistry 420 (1997) 5-9
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E / V vs. Ag/AgCI Fig. 2. (a) Cyclic v o l t a m m o g r a m s o f ca. 70 I.tM m o n o a z a h e m i n reconstituted m y o g l o b i n at an ] n 2 0 3 electrode with high surface hydrophilicity (see text) in a 50 m M bis-Tris buffer solution (pH 6.5) u n d e r N 2 atmosphere at scan rates of 20 to 100 m V s - ~ ; (b) the curve fitting of the v o l t a m m o g r a m obtained at 2 0 m V s ~ after b a c k g r o u n d subtraction with simulated data ( O ) . The digital simulation was m a d e using the values k ¢ ' = 2 . 0 × 1 0 - 4 c m s - 1 , D = 9.0 × 10 -7 cm 2 s i, E ~ = _ 1 8 0 m V vs. Ag[AgCIIKClsat and a (transfer coefficient) = 0.55. Electrode area 0.175 cm 2.
CD spectra of azaMb in oxidized and reduced forms showed that the CD signals at the Soret region were significantly smaller than those of native Mb, although at wavelengths less than 250nm no significant difference in the CD spectrum was observed between azaMb and native Mb, as shown in Fig. 3. The heme CD bands are known [17] to be due to coupled-oscillator interactions between the porphyrin "rr-w * and the "rr--rr ~ transitions in aromatic side chains of near heme residues, including the distal histidine (E7-His). Thus, the CD spectra obtained suggest that the conformation of the heme environment of azaMb changed to some extent, although the coordination
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structure was reported to be an aquomet form [11], which is essentially the same as that of native Mb. Fig. 4(a) shows cyclic voltammograms of hemin and monoazahemin in methanol, where in order to avoid adsorption of reactants onto the electrode a rather fast scan rate (1 V s - l ) was used. The redox potential of monoazahemin shifted positively by ca. 70mV compared with heroin. Again, in the presence of an excess amount of imidazole as axial ligands, the redox potential of monoazaheroin was observed to be more positive than hemin by ca. 120 inV. Similar positive shifts of redox potentials for iron complexes of tetraazaporphyrin by about 400mV after introducing four nitrogen atoms to the porphyrin ring [18] and of phthalocyanines [19] are also known. Such positive shifts of redox potentials have been explained mainly in
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Fig. 3. C D spectra in both (a) oxidized and (b) reduced f o r m s o f ca. 50 p.M ( - - - ) native and ( ) m o n o a z a h e m i n reconstituted m y o globins in a 5 0 r a M bis-Tris buffer solution (pH 6.5). The optical pathlength of the cell was 1 ram.
Fig. 4. Cyclic v o l t a m m o g r a m s of (a) ca. 1001xM ( - - - ) hemin and ( - - ) m o n o a z a h e m i n in m e t h a n o l c o n t a i n i n g 0.1 M NaC104 at a scan rate o f 1 . 0 V s ~ and (b) ca. 501xM ( - - - ) native (or hemin) and ( - - ) m o n o a z a h e m i n reconstituted m y o g l o b i n s at an I n 2 0 3 electrode in a 5 0 r a M bis-Tris buffer solution (pH 6.5) under N 2 atmosphere at a scan rate of 2 0 m V s ~.
8
I. Taniguchi er al./Journal of Electroanalytical Chemistry 420 (1997) 5 - 9
terms of the increased acidity of the ligand by the meso nitrogen (increased electron-withdrawing capacity or "rracceptor character [11,18]), resulting in the larger stabilization of the reduced form of monoazahemin. The present results show that the introduction of only one meso nitrogen to the hemin backbone causes a clear positive shift of redox potentials. Very interestingly, in contrast to the expected positive shift from the redox potential of monoazahemin, the redox potential of azaMb was observed to be more negative than that of native Mb by ca. 40mV (Fig. 4(b)). Since the coordination structure of the redox center of azaMb is confirmed to be the same as that of native Mb (i.e. aquomet form [11 ]), the redox potential observed cannot be explained only by the larger w-acidity of the monoazaheroin ligand compared with hemin, but the heme environment including proximal histidine (F8-His) as the axial ligand must be taken into account to consider its redox potential when monoazahemin is reconstituted inside apoMb. The reason for the more negative redox potential of azaMb than of native Mb is not clear yet, but an induced conformational change at the heme environment of azaMb, as suggested from its CD spectrum (see Fig. 3), would be a main reason. The nature of the sixth ligand affects significantly the redox potential. Fig. 5 shows the voltammograms of
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cyano-myoglobin (CN-Mb) and cyano-monoazahemin reconstituted myoglobin (CN-azaMb). Redox potentials of both cyanide ion substituted myoglobins shifted clearly in the negative direction, because of donated negative charge from cyanide ion to iron. The amount of negative shift in redox potential by introducing the cyanide ion was rather large, about 0.45V from the redox potentials of corresponding aquomet forms for both native and monoazahemin reconstituted myoglobins. Interestingly, moreover, the cyanide ion was bound strongly to the ferrous form of azaMb, and no dissociation of cyanide ions from the reduced azaMb was observed on the timescale used for the cyclic voltammetric measurement (scan rates tested down to 2mVs-~), while the cyanide ion was partly released after reduction of CN-Mb to give a small current for reoxidation of around - 0 . 0 5 V at pH 6.8 (see Fig. 5(a)), due to reoxidation of the produced deoxyMb to metMb. This reoxidation peak shifted a little positively at higher pH (see Fig. 5(a)), because the reversibility of the electrode reaction of Mb became less at higher pH [9]. The present electrochemical observation is in good agreement with the reported kinetic data at pH 7 for cyanide dissociation for azaMb (0.002s -1 [11]) and native Mb (0.14s -I [20]). Also, this behavior is in good agreement with the fact that the dioxygen affinity of reduced azaMb becomes 50 times larger than that of native Mb [11]. Similarly, [3,~-diazamesoporphyrin III (diazahemin), prepared by introduction of two meso nitrogen atoms to a hemin derivative, showed a well-defined voltammogram with its redox potential at - 0 . 1 7 V in methanol (not shown), which is 65mV more positive than the redox potential ( - 0 . 2 3 5 V, see Fig. 4) of monoazahemin. For diazahemin reconstituted myoglobin (diazaMb), however, the redox potential ( - 0 . 1 3 5 V) was close to that of native Mb ( - 0.14 V), shifting positively only 45 mV compared with that of azaMb ( - 0 . 1 7 V , see Fig. 4). The observed redox potential of diazaMb is still more negative than that simply expected from the redox potential of diazahemin itself. This indicates again that the heme environment in the axial direction is primarily important to determine the redox potential of diazahemin reconstituted myoglobin. More details of the electrochemistry of mono- and diazahemin reconstituted myoglobins and their ligand substitution reactions will be discussed later in a separate paper.
E / V vs. Ag/AgCI
Acknowledgements Fig. 5. Cyclic voltammograms of (a) ca. 50 ~M cyano-myoglobin (CNMb) and (b) cyano-monoazahemin reconstituted myoglobin (CN-azaMb) at an In203 electrode in a bis-Tfis buffcr solution in the presence of 33mM NaCN (pH 8.8) under N 2 atmosphere at a scan rate of 2 0 m V s - t, together with the voltammograms of (a) native myoglobin (Mb), cyanomyoglobin (CN-Mb) and (b) monoazahemin reconstituted myoglobin (azaMb) obtained in 50raM bis-Tris buffer solutions at pH 6.8 (for CN-Mb) and 6.5 (for Mb and azaMb).
Partial financial support of this work by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan for Priority Areas (Nos. 07242259 & 08232266) and by the Heiwa Nakashima Foundation is gratefully acknowledged.
L Taniguchi et aL/Journal of Electroanalytical Chemistry 420 (1997) 5-9
References [1] F.A. Schultz and I. Taniguchi (Eds.), Redox Mechanisms and Interfacial Properties of Molecules of Biological Importance, Electrochemical Society, Pennington, NJ, 1993. [2] F.M. Hawkridge and 1. Taniguchi, Comm. lnorg. Chem., 17 (1995) 163. [3] F.A. Armstrong, H.A.O. Hill and N.J. Walton, Acc. Chem. Res., 21 (1988) 407. [4] F.A. Armstrong, Structure and Bonding, Vol. 72, Bioinorganic Chemistry, Springer-Verlag, Berlin, 1990, p. 137. [5] F.A. Armstrong, Perspectives on Bioinorganic Chemistry, Vol. 1, JAI Press, New York, 1991, p. 141. [6] A. Heller, Acc. Chem. Res., 23 (1990) 128. [7] I. Taniguchi, Y. Hirakawa, K. lwakiri, M. Tominaga and K. Nishiyama, J. Chem. Soc., Chem. Commun., (1994) 953. [8] K. Nishiyama, H. lshida and I. Taniguchi, J. Electroanal. Chem., 373 (1994) 255. [9] I. Taniguchi, K. Watanabe, M. Tominaga and F.M. Hawkridge, J. Electroanal. Chem., 333 (1992) 331.
9
[10] M. Tominaga, T. Kumagai, S. Takita and I. Taniguchi, Chem. Lett., (1993) 1771. [1 l] S. Neya, T. Kaku, N. Funasaki, Y. Shiro, T. lizuka, K. Imai and H. Hori, J. Biol. Chem., 270 (1995) 13118. [12] K.D. Hardman, E.H. Eylar, D.K. Ray, L.J. Banaszak and F.R.N. Gurd, J. Biol. Chem., 241 (1966) 432. [13] K.D. Hapner, R.A. Bradshaw, C.R. Hartzell and F.R.N. Gurd, J. Biol. Chem., 243 (1968) 683. [14] F.W.J. Teale, Biochim. Biophys. Acta, 35 (1959) 543. [15] T. Asakura, Methods Enzymol., 52 (1978) 445. [16] M.J. Crumpton and A. Polson, J. Mol. Biol., 11 (1965) 722. [17] M.C. Hsu and R.W. Woody, J. Am. Chem. Soc., 93 (1971) 3515. [18] J.P. Fitzgerald, B.S. Haggerty, A.L. Rheingold, L. May and G.A. Brewer, Inorg. Chem., 31 (1992) 2006. [19] A.B.P. Lever, E.R. Milaeva and G. Speier, in C.C. Leznoff and A.B.P. Lever (Eds.), Phthalocyanines, Vol. 3, VCH, Berlin, 1993, p. 1. [20] A. Bellelli, G. Antonini, M. Brunori, B.A. Springer and S.G. Sligar, J. Biol. Chem., 285 (1990) 18898.
ELSEVIER
Journal of Electroanalytical Chemistry 420 (1997) 5-9
Preliminary note
Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode Isao Taniguchi a,*, Yasuhiro Mie a, Katsuhiko Nishiyama a, Viktor Brabec a,l, Olga Novakova 1 a, Saburo Neya b, Noriaki Funasaki b a Department of Applied Chemistry and Biochemistry. Faculty of Engineering, Kumamoto University, Kurokami, Kumamoto 860, Japan b Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan Received 7 August 1996; revised 30 September 1996
Abstract
Monoazahemin reconstituted myoglobin was prepared and its electrochemical behavior was studied in comparison with native myoglobin. For both myoglobins well-defined voltammograms were clearly obtained at highly hydrophilic surfaces of indium oxide electrodes. Although monoazahemin showed a more positive redox potential than hemin (measured in methanol), monoazahemin reconstituted myoglobin showed a more negative redox potential than native myoglobin in a 50 mM bis-Tris buffer solution (pH 6.5), suggesting that for both native and reconstituted myoglobins the heme environment including proximal histidine as an axial ligand of the redox center plays an important role in determining the redox potential. Also, a unique electrochemical response of cyano-monoazahemin reconstituted myoglobin was demonstrated. Keywords: Myoglobin; Monoazahemin; Reconstituted myoglobin; Indium oxide; Redox potential; Cyclic voltammetry
1. Introduction Recently, electron transfer reactions of metalioproteins at functional electrodes have become one of the most attractive fields in electrochemistry and related fields of chemistry (see for example Ref. [1]), and various electrode reactions of electron transfer proteins have been reported [2-8]. However, electrochemical studies of metalloproteins with biological functions other than electron transfer have still been very limited. Myoglobin (Mb), for example, is a medium-sized heme protein having biological functions of dioxygen storage and transport, and its rapid direct electron transfer at an electrode has been unsuccessful until fairly recently. We have found that Mb from both horse heart and sperm whale skeletal muscle gave well-defined quasi-reversible voltammograms at the highly hydrophilic surface of an In203 electrode [9,10]. Mb is a very attractive biomolecule, not only because of its native physiological functions but also because of the
possible introduction of new artificial functions. The redox center of Mb is located at the heme pocket with no covalent binding to the globin moiety, and therefore can easily be replaced by other molecules to prepare semiartificial myoglobins. In the present study Mb, of which the redox center (hemin) was substituted with the iron complex of ctazamesoporphyrin XIII (monoazahemin), i.e. monoazahemin reconstituted myoglobin (azaMb) [11], has been prepared, and its unique redox responses were observed for the first time in comparison with native metMb. For native and reconstituted myoglobins, the axial ligands of the redox center as well as the porphyrin ring are clearly demonstrated to be important for their electrochemical reactions. For example, although monoazahemin itself has a more positive redox potential than hemin, azaMb showed a more negative redox potential than native Mb.
2. Experimental " Corresponding author. On leave from the Institute of Biophysics, Academy of Sciences, Brno (Czech Republic) as Visiting Research Scholars of the HeiwaNakashima Foundation (VB) and JSPS Research Fellow (ON).
Horse heart Mb was obtained from Sigma, and was purified further by chromatography [12,13] on a Whatman CM-52 column at 4°C using a phosphate buffer solution
0022-0728/97/$17.00 Copyright © 1997 Elsevier Science S.A. All rights reserved. PH S 0 0 2 2 - 0 7 2 8 ( 9 6 ) 0 1 0 2 2 - 4
6
1. Taniguchi et al./Journal of Electroanalytical Chemistry 420 (1997) 5 - 9
(16mM N a H z P O 4 + 3 . 1 m M N%HPO4, pH 6.5; ionic strength /z = ca. 0.025 M) as eluent. The Mb obtained was dialyzed against distilled water and then concentrated by ultrafiltration. The SDS-PAGE measurement of Mb thus obtained gave a single band having an appropriate molecular weight (ca. 17000). Various spectrochemical data showed the sample was native and pure. The concentrations of myoglobin were estimated spectroscopically for ferro Mb (reduced with sodium dithionite) at pH 6.5 using the molar absorptivity of l l 4 0 0 0 M - ~ c m -1 at 434nm [12,13]. Apomyoglobin (apoMb) was prepared from horse heart Mb by a similar procedure to that described in the literature [11] on the basis of the acid methylethylketone method [14,15], and dialyzed against an excess amount of 10mM cold bis-Tris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol) buffer solution (pH 6.5) for more than 24h to remove methylethyiketone. Monoazahemin (see Fig. 1 for its structure) was synthesized according to the literature [l 1]. ApoMb obtained was then mixed in an ice bath with a 1.2 molar equivalent amount of the monoazahemin dissolved in methanol, and dialyzed with an excess amount of the bis-Tris buffer solution overnight to obtain azaMb. AzaMb was then purified by co]umn chromatography on a Whatman CM-52 column below 4°C. The spectrum of the azaMb (Fig. l) was the same as that reported previously [11], and the purity was satisfactory [absorbance ratio A(Soret = 385 nm)/A(280nm) = 3.5, which is smaller than that (5.0) of native Mb]. In the present study, especially for electrochemical use, only fractions having absorbance ratio 3.5 were used. All other reagents used were of analytical grade. 25 to 50mM bis-Tris buffer solutions were used for electro-
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/
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300
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500
600
Wavelength / nm
Fig. I. UV-visible spectra of purified (---) native myoglobin, ( ) monoazahemin reconstituted myoglobin (in ferric forms) and ( - - - ) apomyoglobinin a 50raM bis-Tris buffer solution (pH 6.5), together with the structures of heroin and monoazahemin as prosthetic groups.
chemical measurements, where the electrochemical responses were independent of the buffer concentrations. Cyclic voitammograms were obtained at 25°C using a BAS CV-50W electrochemical system at an In20 3 electrode (approximately 5 X 5 m m 2, from Kinoene Optics Corp., Japan) under nitrogen atmosphere. The electrode was cleaned by ultrasonication in a 1% aqueous New-Vista (anionic surfactant, AIC Corp.) solution, followed by ultrasonic washing in distilled water until the surface of the electrode became fully hydrophilic. The surface hydrophilicity was monitored by measuring the surface tension of the electrode by the Wilhelmy method using a Shimadzu ST-I surface tensometer [10] to be more than 7 0 d y n c m -t at 25°C in water (cf. the surface tension of water itself at 25°C, equal to 72 dyn c m - a). A Pt plate and an AglAgCIIKC1sat were used as the counter and reference electrodes respectively. The UV-visible and circular dichroism (CD) spectra were measured using Shimadzu UV-2100 and JASCO J-720 spectrophotometers respectively.
3. Results and discussion
For metMb electrochemistry, the surface hydrophilicity of an In203 electrode affected very much the peak separation of the voltammogram, and almost fully hydrophilic surfaces ( > 70 dyn c m - ~) of In203 electrodes were useful in giving well-defined voltammograms for both sperm whale skeletal muscle and horse heart metMb with a heterogeneous electron transfer rate constant k ~' of ca. (1 to 3 ) × 10 -4 c m s - ~ at pH 6.5, as reported previously [9,10]. Similarly, for azaMb, well-defined voltammograms were obtained at a highly hydrophilic surface of In203 in a 50 mM bis-Tris buffer solution (pH 6.5) as shown in Fig. 2(a). No significant change in the voltammograms was observed during the continuous measurements for at least an hour. The peak current was linear with the concentration of the azaMb tested (up to ca. 0.1 mM), and was proportional to the square root of the scan rate, u ~/2, in the u region up to 100mVs-~. At 25°C the diffusion coefficient D of azaMb, estimated by both cyclic voltammetry and potential-step chronocoulometry after correction of the active area of the electrode surface from the geometric one, was ca. 9.0( _ 1.0) × 10- 7 cm 2 s- l, which is in good agreement with that reported for sperm whale Mb (1.1 × 10 -6 cm 2 s-1 [16]). The formal redox potential E ~ was evaluated, as the midpoint of anodic and cathodic peak potentials of the redox wave, to be - 0 . 1 8 V vs. Ag [AgCIIKCIsa t, which is more negative than that of native Mb by ca. 40mV. This point will be discussed in more detail later in the paper. The observed voltammogram showed that the reaction was quasi-reversible, and the k~' value of ca. 2 x 10 4 cm s- ~ was estimated, using a digital simulation technique (see Fig. 2(b)), to be similar to that of native Mb [9,10].
I. Taniguchi et al./ Journal of Electroanalytical Chemistry 420 (1997) 5-9
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E / V vs. Ag/AgCI Fig. 2. (a) Cyclic v o l t a m m o g r a m s o f ca. 70 I.tM m o n o a z a h e m i n reconstituted m y o g l o b i n at an ] n 2 0 3 electrode with high surface hydrophilicity (see text) in a 50 m M bis-Tris buffer solution (pH 6.5) u n d e r N 2 atmosphere at scan rates of 20 to 100 m V s - ~ ; (b) the curve fitting of the v o l t a m m o g r a m obtained at 2 0 m V s ~ after b a c k g r o u n d subtraction with simulated data ( O ) . The digital simulation was m a d e using the values k ¢ ' = 2 . 0 × 1 0 - 4 c m s - 1 , D = 9.0 × 10 -7 cm 2 s i, E ~ = _ 1 8 0 m V vs. Ag[AgCIIKClsat and a (transfer coefficient) = 0.55. Electrode area 0.175 cm 2.
CD spectra of azaMb in oxidized and reduced forms showed that the CD signals at the Soret region were significantly smaller than those of native Mb, although at wavelengths less than 250nm no significant difference in the CD spectrum was observed between azaMb and native Mb, as shown in Fig. 3. The heme CD bands are known [17] to be due to coupled-oscillator interactions between the porphyrin "rr-w * and the "rr--rr ~ transitions in aromatic side chains of near heme residues, including the distal histidine (E7-His). Thus, the CD spectra obtained suggest that the conformation of the heme environment of azaMb changed to some extent, although the coordination
a) x 106
x 105
10
I
I
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structure was reported to be an aquomet form [11], which is essentially the same as that of native Mb. Fig. 4(a) shows cyclic voltammograms of hemin and monoazahemin in methanol, where in order to avoid adsorption of reactants onto the electrode a rather fast scan rate (1 V s - l ) was used. The redox potential of monoazahemin shifted positively by ca. 70mV compared with heroin. Again, in the presence of an excess amount of imidazole as axial ligands, the redox potential of monoazaheroin was observed to be more positive than hemin by ca. 120 inV. Similar positive shifts of redox potentials for iron complexes of tetraazaporphyrin by about 400mV after introducing four nitrogen atoms to the porphyrin ring [18] and of phthalocyanines [19] are also known. Such positive shifts of redox potentials have been explained mainly in
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.
.
.
.
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E / V vs. Ag / AgCI
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Fig. 3. C D spectra in both (a) oxidized and (b) reduced f o r m s o f ca. 50 p.M ( - - - ) native and ( ) m o n o a z a h e m i n reconstituted m y o globins in a 5 0 r a M bis-Tris buffer solution (pH 6.5). The optical pathlength of the cell was 1 ram.
Fig. 4. Cyclic v o l t a m m o g r a m s of (a) ca. 1001xM ( - - - ) hemin and ( - - ) m o n o a z a h e m i n in m e t h a n o l c o n t a i n i n g 0.1 M NaC104 at a scan rate o f 1 . 0 V s ~ and (b) ca. 501xM ( - - - ) native (or hemin) and ( - - ) m o n o a z a h e m i n reconstituted m y o g l o b i n s at an I n 2 0 3 electrode in a 5 0 r a M bis-Tris buffer solution (pH 6.5) under N 2 atmosphere at a scan rate of 2 0 m V s ~.
8
I. Taniguchi er al./Journal of Electroanalytical Chemistry 420 (1997) 5 - 9
terms of the increased acidity of the ligand by the meso nitrogen (increased electron-withdrawing capacity or "rracceptor character [11,18]), resulting in the larger stabilization of the reduced form of monoazahemin. The present results show that the introduction of only one meso nitrogen to the hemin backbone causes a clear positive shift of redox potentials. Very interestingly, in contrast to the expected positive shift from the redox potential of monoazahemin, the redox potential of azaMb was observed to be more negative than that of native Mb by ca. 40mV (Fig. 4(b)). Since the coordination structure of the redox center of azaMb is confirmed to be the same as that of native Mb (i.e. aquomet form [11 ]), the redox potential observed cannot be explained only by the larger w-acidity of the monoazaheroin ligand compared with hemin, but the heme environment including proximal histidine (F8-His) as the axial ligand must be taken into account to consider its redox potential when monoazahemin is reconstituted inside apoMb. The reason for the more negative redox potential of azaMb than of native Mb is not clear yet, but an induced conformational change at the heme environment of azaMb, as suggested from its CD spectrum (see Fig. 3), would be a main reason. The nature of the sixth ligand affects significantly the redox potential. Fig. 5 shows the voltammograms of
(a) m b {pH 6.5) 0.2p.A
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./~\.-. 3
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cyano-myoglobin (CN-Mb) and cyano-monoazahemin reconstituted myoglobin (CN-azaMb). Redox potentials of both cyanide ion substituted myoglobins shifted clearly in the negative direction, because of donated negative charge from cyanide ion to iron. The amount of negative shift in redox potential by introducing the cyanide ion was rather large, about 0.45V from the redox potentials of corresponding aquomet forms for both native and monoazahemin reconstituted myoglobins. Interestingly, moreover, the cyanide ion was bound strongly to the ferrous form of azaMb, and no dissociation of cyanide ions from the reduced azaMb was observed on the timescale used for the cyclic voltammetric measurement (scan rates tested down to 2mVs-~), while the cyanide ion was partly released after reduction of CN-Mb to give a small current for reoxidation of around - 0 . 0 5 V at pH 6.8 (see Fig. 5(a)), due to reoxidation of the produced deoxyMb to metMb. This reoxidation peak shifted a little positively at higher pH (see Fig. 5(a)), because the reversibility of the electrode reaction of Mb became less at higher pH [9]. The present electrochemical observation is in good agreement with the reported kinetic data at pH 7 for cyanide dissociation for azaMb (0.002s -1 [11]) and native Mb (0.14s -I [20]). Also, this behavior is in good agreement with the fact that the dioxygen affinity of reduced azaMb becomes 50 times larger than that of native Mb [11]. Similarly, [3,~-diazamesoporphyrin III (diazahemin), prepared by introduction of two meso nitrogen atoms to a hemin derivative, showed a well-defined voltammogram with its redox potential at - 0 . 1 7 V in methanol (not shown), which is 65mV more positive than the redox potential ( - 0 . 2 3 5 V, see Fig. 4) of monoazahemin. For diazahemin reconstituted myoglobin (diazaMb), however, the redox potential ( - 0 . 1 3 5 V) was close to that of native Mb ( - 0.14 V), shifting positively only 45 mV compared with that of azaMb ( - 0 . 1 7 V , see Fig. 4). The observed redox potential of diazaMb is still more negative than that simply expected from the redox potential of diazahemin itself. This indicates again that the heme environment in the axial direction is primarily important to determine the redox potential of diazahemin reconstituted myoglobin. More details of the electrochemistry of mono- and diazahemin reconstituted myoglobins and their ligand substitution reactions will be discussed later in a separate paper.
E / V vs. Ag/AgCI
Acknowledgements Fig. 5. Cyclic voltammograms of (a) ca. 50 ~M cyano-myoglobin (CNMb) and (b) cyano-monoazahemin reconstituted myoglobin (CN-azaMb) at an In203 electrode in a bis-Tfis buffcr solution in the presence of 33mM NaCN (pH 8.8) under N 2 atmosphere at a scan rate of 2 0 m V s - t, together with the voltammograms of (a) native myoglobin (Mb), cyanomyoglobin (CN-Mb) and (b) monoazahemin reconstituted myoglobin (azaMb) obtained in 50raM bis-Tris buffer solutions at pH 6.8 (for CN-Mb) and 6.5 (for Mb and azaMb).
Partial financial support of this work by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan for Priority Areas (Nos. 07242259 & 08232266) and by the Heiwa Nakashima Foundation is gratefully acknowledged.
L Taniguchi et aL/Journal of Electroanalytical Chemistry 420 (1997) 5-9
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[10] M. Tominaga, T. Kumagai, S. Takita and I. Taniguchi, Chem. Lett., (1993) 1771. [1 l] S. Neya, T. Kaku, N. Funasaki, Y. Shiro, T. lizuka, K. Imai and H. Hori, J. Biol. Chem., 270 (1995) 13118. [12] K.D. Hardman, E.H. Eylar, D.K. Ray, L.J. Banaszak and F.R.N. Gurd, J. Biol. Chem., 241 (1966) 432. [13] K.D. Hapner, R.A. Bradshaw, C.R. Hartzell and F.R.N. Gurd, J. Biol. Chem., 243 (1968) 683. [14] F.W.J. Teale, Biochim. Biophys. Acta, 35 (1959) 543. [15] T. Asakura, Methods Enzymol., 52 (1978) 445. [16] M.J. Crumpton and A. Polson, J. Mol. Biol., 11 (1965) 722. [17] M.C. Hsu and R.W. Woody, J. Am. Chem. Soc., 93 (1971) 3515. [18] J.P. Fitzgerald, B.S. Haggerty, A.L. Rheingold, L. May and G.A. Brewer, Inorg. Chem., 31 (1992) 2006. [19] A.B.P. Lever, E.R. Milaeva and G. Speier, in C.C. Leznoff and A.B.P. Lever (Eds.), Phthalocyanines, Vol. 3, VCH, Berlin, 1993, p. 1. [20] A. Bellelli, G. Antonini, M. Brunori, B.A. Springer and S.G. Sligar, J. Biol. Chem., 285 (1990) 18898.