Etiohemin as a prosthetic group of myoglobin

Biochimica et Biophysica Acta, 996 (1989) 226-232 Elsevier

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Etiohemin as a prosthetic group of myoglobin S a b u r o N e y a ~, N o r i a k i F u n a s a k i ~ a n d K i y o h i r o Imai 2 Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina and z Department of PhysicochemicalPhysiology, Medical School, Osaka University, Osaka (Japan)

(Received 17 January 1989)

Key words: Etioheme; Myoglobin; Coordination structure; Heme recognition; Oxygen binding

Sperm whale myoglobin was reconstituted with etioheme and the stoichiometric complex formation was confirmed. The proton NMR spectrum of the deoxy myoglobin exhibits an NH signal from the proximal histidine at 78.6 ppm, indicating heine incorporation into the heine pocket to form the Fe-N(His-F8) bond. The appearance of a single set of the heine-methyl NMR signals shows that etioheme without acid side-chains specifically interacts with the surrounding globin. The visible spectral data suggest retention of a normal iron coordination structure. The functional and NMR spectral properties of etioheme myoglobin are similar to those of mesoheme myoglobin, reflecting the absence of the electron-withdrawing heine vinyl groups.

Introduction Myoglobin is an oxygen storage protein containing protoheme as the prosthetic group. The polypeptide chain prevents irreversible oxidation of the iron. Since simple ferrous heme cannot reversibly bind oxygen at room temperature, the hydrophobic interactions between the heme and globin are required for normal function. The heme-globin interactions have been examined by removal of the protoheme followed by reconstitution with modified hemes. Many of the heroes employed are protoheme analogs [1-6]. Unlike the natural hemes, etioheme lacks propionyl groups and it is an all-alkyl side-chain compound [7]. Coupling of etioheme with apoglobin was first reported by O'Hagan [8] and O'Hagan and George [9]. The etioheme used was obtained by decarboxylation of mesoheme [10]. The protein reconstitution with etioheme was extended by several other workers [11,12]. Although this pioneer work suggested the binding of etioheme with globin, the structure, absorption spectra and function of the reconstituted proteins are only partially characterized. Etioheme has notable structural features. Each pyrrole ring bears a methyl group, like protoheme. The type III isomer derived from mesoheme [10], however, is

not symmetric about the diagonal meso-carbon axis. Recent spectroscopic studies have demonstrated that serious heme rotational disorder occurs when globin is reconstituted with an asymmetric heme about the meso-carbon axis [13-16]. It was further demonstrated that several alkyl hemes rotate about the Fe-N(His-F8) bond in the heme pocket [17,18]. The molecular structure of etioheme suggests the possibility that several isomers rotate about the meso-carbon axis and/or that the Fe-N(His-Fg) bond may exist in the reconstitu~.ed protein. Another characteristic of etioheme is the equivalence of the constituent pyrrole rings. All of them are equally substituted with methyl and ethyl groups. This is in contrast with protoheme, having two types of pyrrole. In protoheme, the two pyrroles have methyl and vinyl residues and the other two are substituted with methyl and propionyl side-chains Equivalence of the pyrroles in etioheme must affect the heme electronic structure of the reconstituted protein, thougn such influences have not so far been elucidated. We have attempted to solve the above problems in our present study on the basis of detailed structural and functional characterization of etioheme-reconstituted myoglobin. We used etioheme I (Fig. 1), rather than the type III isomer [10], because the type I isomer is prepared in a large quantity from the pyrrole precursor [19].

Abbrevia*ions: etioheme, the iron complex of 1,3,5,7-tetraethyl2,4,6,8-tetramethylporphyrin; DSS, sodium 2,2-dimethyl 2-silapentane-5-sulfonate.

Materials and Methods

Correspondence: S. Neya, Department of Physical Chemistry, Kynto Pharmaceutical University, Yamashina, Kyoto 607, Japan.

Reconstituted myoglobin Etioporphyrin (type I isomer) was synthesized by the literature methods [19], and iron was incorporated by

0167-4838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

227 refluxing the heme with FeCI 2 in dimethylformamide [20]. Sperm whale myoglo~in (type II) was purchased from Sigma. Apoglobin prepared from 0.1 g of myoglobin was combined with a 2-fold molar" excess (about 7 nag) of ferric etioheme chloride according to the reported procedures [21,22]. The crude mixture was dialyzed against several changes of 10 mM Bistris buffer (pH 6.0), 40C, for 12-16 h and loaded on a carboxymethylated-cellulose column equilibrated with the same buffer. The purified myoglobin was eluted from the column with a linear gradient of Tris buffer, 10-100 mM (each 400 ml), at pH 7.0 and 4 ° C. Fractions with an absorbance ratio A393nm/A2sonmof 6.0 or larger were collected. Ferric myoglobin derivatives were obtained by adding ligand stock solution in 0.1 M Tris buffer (pH 7.0). Ferrous complexes were prepared by reduction of the ferric protein with Na 2S204 .

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Spectrophotometric measurements Electronic absorption spectra were recorded with a Shimadzu MPS-2000 spectrophotometer equipped with a thermostatted sample compartment. Proton NMR spectra were obtained at 300 MHz on a Varian XL-300 spectrometer as described previously [17]. The solvent exchange from H20 to 1320 for the NMR observation was performed with an Amicon membrane, PM-10. The pD values of NMR samples were the direct reading of a pH meter Toko-Kagaku, TP-1000.

Pyridine hemochromogen A weighed amount of ferric etioheme chloride was dissolved into pyridine and the solution was diluted with 1:1 (v/v) pyridine/dimethylformamide. The reduced hemochromogen was prepared by adding a small volume (about 20/~1) of aqueous Na2S204 to 4.0 ml of the heine solution. Absorption maxima of the ferrous hemochromogen were located at 406 (111 mM -~. era-l), 514 (17.1), and 544 (30.4) nm. On the basis of these values, the visible parameters of etioheme-reconstituted myoglobin were determined.

Fig. 1. Structure of the etioheme employed in the present work. This is the type I compound of the four possible type-isomers.

pK s determination The pK 3 of etioporphyrin was spectrophotometrically determined with the method used for octaethylporphyrin [17]. Analysis of the pH-dependent absorption at 388 nm and 20 °C yielded a pK 3 of 6.38 + 0.11 in 2.5% sodium dodecyl sulfate.

Ligand binding study The equilibrium ligand-binding constants were determined by spectrophotometric titrations. The oxygenbinding curve was recorded with Imai's automatic recording apparatus [23] in 0.1 M phosphate (pH 7.4), 20 o C, iL .he presence of the reducing enzyme system I241. Results

Spectral properties The spectrophotometric titration of etioheme with apomyoglobin showed their 1:1 bh~ding stoichiometry (Fig. 2). The purified metmyoglobin exhibits a sharp Sorer peak at 393 nm. The absorbance ratio A393nm/A 280 nm was constantly 6.2 for several preparations. The large ratio indicates high purity of the reconstituted myoglobin. Etioheme metmyoglobin binds several anionic ligands. The affinities for CN-, N 3 , imidazole, SCN-, and F - were 2.4-l0 s, 5.2.104, 2.1.103, 2.5.10 4, and 88 M - : ; respectively, in 0.1 M Tris (pH 7.0), 20oC. These are comparable with the ligand affinities reported for native myoglobin [25]. The ferrous complexes are readily derived from the metmyoglobin. Tile visible absorption spectra of the ferric and ferrous complexes are indicated in Fig. 3, and the spectral data arc summarized in Table I. The absorption m~xima are blueshifted by about 15 nm as compared with those of the native protein [26]. The overall features are similar to those of octaethylheme-substituted myoglobin [17]. The

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visible absorption of the metmyoglobin markedly changes with pH (Fig. 4). From the analysis of the 566 nm transition, an apparent pK, of 8.35 + 0.23 was obtained. Among the paramagnetic resonances, the heme methyl peaks are especially useful because they directly reflect subtle changes of the iron electronic state and because the prominent peaks are easily identified in the NMR spectra [27,28]. Etioheme, like natural hemes, has four methyl groups attached to the pyrrole rings (Fig. 1). In Fig. 5A, the NMR spectra of ferric low-spin compounds are indicated. The heme methyl signals are observed at 24.6, 21.4, 16.4 and 12.1 ppm for the cyanide complex. The 12.1 ppm peak appears to be

TABLE •

larger than other methyl signals, due to coincidental overlap of minor peaks. The heme methyl peaks of the azide and imidazole complexes are also labeled with their shifts. The temperature dependence of the heme methyl sY:fts of the cyanide complex obeys the Curie law (Fig. 5B). In the spectra of the high-spin metmyoglobin (Fig. 6), the heine methyl signals are also labeled with the chemical shifts. Some of the heme methyl signal,~ overlap with each other to give relatively large-intensity signals. The average heme methyl shift of the aquomet myogiobin, 75.6 ppm, is in agreement with 75.8 ppm reported for native myoglobin [29]. The proton NMR spectrum of the deoxy derivative recorded in H20 solvent is shown in Fig. 7. We note an exchangeable signal at 78.6 ppm. The peak position is comparable with that of the NH peaks of the proximal F8 histidine of several myoglobins [17,28,30].

Visible absorption parameters of etioheme myoglobin

lmid., imidazole.

Ligand

Ferric H,O SCN-

Spin equilibrium analysis

h(¢) a

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525 (9.8) 530 (9.5) 530 (9.6)

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543 (11.2) 531 (13.0) 527 (13.2)

566 (13.1) 555 (11.0)

a Wavelength in nm and absorption coefficient in mM-~. c m - ! in 0.1 M Tris at pH 7.0 and 20 o C.

The azide complexes of ferric hemoproteins are in thermal equilibrium between the high- (S = 5/2) and low-spin (S = 1/2) states [31,32]. The equilibrium position is primarily determined with the ligand-field strength of the iron [33,34]. We analyzed the spin equilibrium of the azide complex of etioheme myoglobin to examine the Fe-N(His-F8) interaction. The equilibrium is reflected in the NMR spectra as abnormal temperature-dependence of the isotropic shift (Fig. 8). In the Curie plot of the methyl signals, the temperaturedependence of the peaks I and 2 is rather small and the slope of the signals 3 and 4 is opposite in sign to that expected from the Curie law. The anomalies were

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Fig. 4. (A) Acid-alkaline transition of etioheme metmyoglobin in 5 mM Tris at 20 o C. The pH increases from 7.00 to 7.72, 8.05, 8.29, 8.54, 8.87, 9.00, 9.22 and 9.84 as indicated by the arrows. Heme concentration was 55/tM. (B) pH dependence of the absorbance at 566 nm. The solid curve is the theoretical fit of a single proton equilibrium with pK a = 8.35.

analyzed with the van't Hoff equation derived for the heine methyl signal:

intervals a n d all their possible c o m b i n a t i o n s were c h e c k e d with the aid of a m i c r o c o m p u t e r [35]. W h e n we a n a l y z e d the shifts of the m e t h y l peak 1, the ' b e s t ' l i n e a r correlation was o b t a i n e d at H - - 12 850 a n d L 6850 p p m . K, with a c o r r e l a t i o n coefficient of 0,999865. W i t h these fitting p a r a m e t e r s , A H = - 3 2 7 0 =!= 50 cal/mol and AS=-9.05 + 0.18 e.u. were o b t a i n e d . O t h e r ( H , L ) sets for the p e a k s 2, 3 a n d 4 were (11 900, 6200), (14300, - 1 4 0 0 ) , a n d (13250, -1850), respectively. T h e solid c u r v e s ' i n Fig. 8, calculated w i t h those p a r a m e t e r s , reflect well the observation suggesting the

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Fig. 5. (A) Hyperfine-slfifted proton NMR spectra of etioheme metmyoglobin in the low-spin state. The spectra were recorded in 0.1 M Tris at pD 7.0 and 22°C. Heme concentration was 2-4 mM. The heine methyl signals are labeled with their chemical shifts. (B) The Curie plots for the cyanmet myoglobin in a 10-60 °C range.

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Although etioheme myoglobin reversibly binds oxygen, its functional property has not yet been reported. We measured an oxygen equilibrium curve. The precise Hill plots in Fig. 9 have a slope of 1.02. The partial oxygen pressure at half saturation was 0.60 mmHg. Discussion

validity of the analysis. T h e low-spin fraction of the a z i d e m e t m y o g l o b i n was 0.74:1:0.03 at 20 ° C. T h e temperature at which the high- and low-spin p o p u l a t i o n s are the s a m e was calculated to be 362 + 13 K.

Coordination environment C o u p l i n g o f e t i o h e m e w i t h a p o m y o g l o b i n was origin a l l y a s s u m e d b y O ' H a g a n [8]. T h e p r e s e n t s p e c t r o -

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Fig. 8 (A) Temperature-dependent NMR spectra of azidemet myoglobin reconstituted with etioheme. Peaks 1 to 4 denote the ~eme methyl signals. Peaks 3 and 4 are shifted into the diamagnetic envelope below 5 o C. The spectra were recorded in 0.1 M Tris (pD 7.0) c o n t ~ n g 50 mM NaN 3. (B) Curie plots of the heine methyl signals. The s31id curves are a theoretical fit calculated with the parameters described in the text.

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In view of the structural similarity, the normal /'5o value of etioheme (0.60 mmHg) myoglobin appears reasonable. The slightly higher oxygen affinity of etioheme myoglobin, as compared with the native protein (/'5o = 1.0 mmHg [36]), may reflect increased basicity of the heme nitrogen [37] (pK 3 = 6.4 for etioporphyrin vs. 4.8 [38] for protoporphyrin).

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phosphate(pH 7.4),25oC. The slope and oxygenaffinityare n = 1.02 and Ps0 = 0.60 mmHg, respectively. Y, fractional saturation of myoglobinwithoxygen: P, partial oxygenpressurein mmHg.

scopic and ligand-bin6ing results support his assumption. The direct evidence for etioheme incorporation into the heine pocket comes from the NMR spectrum of the deoxy myoglobin (Fig. 7). Appearance of a single peak at 78.5 ppm demonstrates the high specificity of the iron-globin ir,teraction. From the NMR spectral analogy of several myoglobins [17,28~30], the 78.5 ppm line is assigned to the proximal F8 histidine of etioheme-bound myoglobin. Since this sizable downfield shift is induced by delocalization of iron spin through the Fe-N(His-FS) bond [28], the signal provides confirmative evidence of heme incorporation into the heme cavity to form the Fe-N(His-FS) bond. The blue-shifted visible maxima of the reconstituted myoglobin (Table I) are consistent with an increased ability of the iron to accept electron density from the heme nitrogens [36] due to the absence of electron-withdrawing side-chains in etioheme. The pH-dependent visible spectrum of Fig. 4 indicates the presence of an iron-coordinated water molecule. We can refer to the Fe-N(His-F8) bonding from the spin equilibrium analysis of the azide complex. Since the equilibrium is primarily modulated by the Fe-N (His-F8) interaction [33,34], the similarity in thermodynamic parameters of etioheme-substituted (Fig. 8) and native [32] azidemet myoglobins indicates comparable Fe-N(His-FS) interactions. The exchangeable NH signal of deoxy myoglobin is another probe for the Fe-B(His-F8) bonding [28]. Deoxyetioheme-myoglobin exhibits the signal at 78.6 ppm (Fig. 7), in agreement with 77.8 ppm of native myoglobin [28]. The above results suggest that the normal iron coordination state is conserved after substitution of etioheme for protoheme.

Heme recognition in the myoglobin Recent spectroscopic work has revealed that the protein reconstitution with asymmetric heme induces serious heme disorder with a reversed heme-orientationa! isomer [13-16]. Since etioheme is asymmetric about the diagonal meso-carbon axis (Fig. !), heine disorder is possible. The proton NMR spectra of etioheme-substituted myoglobin derivatives in Figs. 5 and 6 show only one set of heme methyl resonances. These results are interpreted to indicate that the incorporated heme adopts a single dominant orientation. The apparent absence of etioheme disorder is explained by a rapid equilibration process in the heme coupling [14]. Etioheme lacks acid side-chains. The propionyl groups of protoheme in myoglobin are extended to the globin surface to interact with solvent and charged residues of myoglobin [39]. The propionyl residues appear to define the orientation of the inserted protoheme. Since the stabilization by propionyl groups is absent in etioheme myoglobin, etioheme in the heme pocket may be mobile. Indeed, recent NMR work has demonstrated the rotational motion of alkyl porphyrin about the Fe-N(His-F8) bond [17,18]. Since the heine methyl shift of the ferric low-spin heme is sensitive to the orientation of the proximal imidazole [40], the low-spin NMR spectrum provides a clue for the rotational isomerization about the heme normal. Fig. 5A suggests that a single set of the four heme methyls is observed for each of the low-spin derivatives, indicating a single dominant etioheme orientation relative to the Fe-N(His-F8) bond. The normal Curie-type behavior of the heine methyl signals (Fig. 5B) further indicates that that heme rotation about the Fe-N(His-F8) linkage does not occur up to 60°C. From the above results it can be concluded that the incorporated heme is likely to be recognized with high specific preference by the globin. Similarity with mesoheme Etioheme is a typical alkylheme, and allows us to evaluate the role of the electron-withdrawing heme side-chains of natural hemin. The heine methyl shifts in paramagnetic proton NMR spectrum is sensitive to the local electronic states of the four pyrrole rings [40,41]. The equivalence of the pyrrole rings of etioheme pyrroles in myoglobin is reflected in the NMR spectrum as significantly reduced heine methyl dispersion. When we compared etioheme aquomet-myoglobin (Fig. 6) with

232 native myoglobin [29], the signal sep:~ation of the former (22.9 ppm) is about half of the latter (38.5 ppm). A similar result is obtained for the low-spin complexes (Fig. 5 vs. Fig. 2 of Ref. 29). The decreased heine-methyl separation of etioheme myoglobin primarily reflects increased equivalence of the four pyrroles arising from the absence of the electron-withdrawing heme side-chains in etioporphyrin. It is to be noted that the heine methyl spread of etioheme aquomet-m)oglobin 22.9 ppm is comparable with 25.4 ppm of mesoheme substituted myoglobin with an oppositely oriented heine [14]. For the low-spin azidemet complexes, the signal separations are 20.8 and 19.0 ppm for the etioheme (Fig. 5) and mesoheme- (Neya and Funasaki, unpublished data) substituted-myoglobins, respectively. From this point of view, the influence of the 6,7-propionyl side-chains in modulating the iron spin distribution appears relatively small, and etioheme is regarded as a close analog of mesoheme. The similarity of the/'50 value of etioheme myoglobin with that of mesoheme myoglobin (/'50 - 0.4 mmHg [37]) is consistent with this idea. Acknowledgment We are grateful to Ms. Matsuko Fujii for her skillful assistance in preparing etioporphyrin. References 1 DiNello, R.K. and Dolphin, D.H. (1981) J. Biol. Chem. 256, 6903-6912. 2 Kawabe, K., Imaizumi, K., lmai, K., Tyuma, I., Ogoshi, H. and Yoshida, Z, (1982) J. Biochem. (Tokyo) 92, 1703-1712. 3 Makino, R. and Yamazaki, i. (1974) Arch. Biochem. Biophys. 165, 485-493. 4 Seybert, D.W., Moffat, K., Gibson, Q.H. and Chang, C.K. (1977) J. Biol. Chem. 252, 4225-4231. 5 Sono, M. and Asakura, T. (1974) Biochemistry 13, 4386-4394. 6 Tamura, M., Asakura, T. and Yonetani, I'. (1973) Biochim. Biophys. Acta 295, 467-479. 7 Smith, K.M. (1975) in Porphyrins and Metalloporphyrins, pp. 3-$8, Elsevier, Amsterdam. 80'Hagan, J.E. (1960) Biochem. J. 74, 417-423. 90'Hagan, £E. and George, P. (1960) Biocl'~.m.J. 74, 424-427.

10 Shumm, O. (1929) Hoppe-Seyler's Z. Physiol. Chem. 181, 141-175. 11 Asakura, T., Yoshikawa, H. and Imahori, K. (168) J. Biochem. (Tokyo) 64, 515-520. 12 Sugita, Y. and Yoneyama, Y. (1971) J. Biol. Chem. 246, 389-394. 13 La Mar, G.N., Budd, D.L., Smith, K. and Langry, K.C. (1978) Proc. Natl. Acad. Sci. USA 75, 5755-5759. 14 La Mar, G.N., Toi, H. and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401. 15 Santucei, R., Mintrovitch, J., Constantindis, I., Sattedee, J.D. and Ascoli, F. (1988) Biochim. Biophys. Acta 953, 201-204. 16 Gersonde, K., Yu, N.-T., Kerr, E.A., Smith, K.M. and Parish, D.W. (1987) J. Mol. Biol. 194, 545-556. 17 Neya, S. and Funasaki, N. (1987) J. Biol. Chem. 262, 6725-6728. 18 Neya, S., Funasaki, N. and Imai, K. (1988) J. Biol. Chem. 263, 8810-8815. 19 Fuhrhop, J.-H. and Smith, K.M. (1975) in Porphyrins and Metalloporphyrins, pp. 757-869, Elsevier, Amsterdam. 20 Adler, A.D., Longo, F.R., Kampas, F. and Kim, J. (1970) J. Inorg. Nucl. Chem. 32, 2443-2445. 21 Asakura, T. (1978) Methods Enzymol. 52, 456-463. 22 Asakura, T. and Yonetani, T. (1969) J. Biol. Chem. 244, 4573-4579. 23 lmai, K. (1981) Methods Enzymol. 76, 438-449. 24 Hayashi, A., Suzuki, T. and Shin, M. (1973) Biochim. Biophys. Acta 310, 309-316. 25 Sono, M. and Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502. 26 Brill, A.S. and Williams, R.J.P. (1961) Biochem. J. 78, 246-253. 27 WiRhrich, K. (1970) Struct. Bonding 8, 53-121. 28 La Mar, G.N., Budd, D.L. and Goff, H. (1977) Biochem. Biophys. Res. Commun. 77, 104-110. 29 La Mar, G.N., Budd, D.L. and Smith, K.M. (1980) Biochim. Biophys. Acta 622, 210-218. 30 Neya, S. and Funasaki, N. (1988) Biochim. Biophys. Acta 952, 150-157. 31 Beetlestone, J. and George, P. (1964) Biochemistry 3, 707-714. 32 Iizuka, T. and Yonetani, T. (1970) Adv. Biophys. 1, 438-449. 33 Scheidt, W.R., Lee, Y.J., Geiger, D.K., Taylor, K. and Hatano, K. (1982) J. Am. Chem. Soc. 104, 3367-3374. 34 Neya, S., Hada, S., Funasaki, N., Umemura, J. and Takenaka, T. (1985) Biochim. Biophys. Acta 827, 157-163. 35 Neya, S. and Funasaki, N. (1986) Biochemistry 25, 1221-1226. 36 Sono, M. and Asakura, T. (1976) J. Biol. Chem. 251, 2664-2670. 37 Sono, M. and Asakura, T. (1975) J. Biol. Chem. 250, 5227-5232. 38 Falk, J.E. (1966) in Porphyrins and Metalloporphyrins, pp. 26-29, Elsevier, Amsterdam. 39 Takano, T. (1977) J. Mol. Biol. 110, 537-568. 40 Shulman, R.G., Glarum, S.H. and Karplus, M. (1971) J. Mol. Biol. 57, 93-115. 41 La Mar, G.N., Viscio, D.B., Smith, K.M., Caughey, W.S. and Smith, M.L. (1978) J. Am. Chem. Soc. 100, 8085-8092.