©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel Ligand Binding Properties of the Myoglobin Substituted with Monoazahemin (*)

Saburo Neya (§) , Tomoko Kaku , Noriaki Funasaki , Yoshitsugu Shiro (1), Tetsutaro Iizuka (1), Kiyohiro Imai (2), Hiroshi Hori (3)

From the (1) Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, the Institute of Physical and Chemical Research (RIKEN), Saitama 351-01, the (2) Department of Physicochemical Physiology, Medical School, Osaka University, Suita, Osaka 565, and the (3) Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The iron complex of -azamesoporphyrin XIII was combined with apomyoglobin to investigate influence of the meso nitrogen on ligand binding properties in the reconstituted protein. Stoichiometric complex formation between the two components was confirmed, and conservation of the native coordination structures in the resultant myoglobin was established with spectroscopic criteria and apparently normal ligand binding. The visible absorption spectra of various ferric and ferrous derivatives are characteristic with less intense Soret peaks and enhanced visible bands. The electron paramagnetic resonance spectrum with g = 5.2 suggests an anomalous intermediate spin (S = 3/2) character for the aquomet protein. The oxygen affinity of reduced azaheme myoglobin, 0.010 mm Hg, is 50 times larger than that of the native myoglobin. In addition, azaheme myoglobin forms stable complexes with imidazole, pyridine, or cyanide in ferrous state. All of these new properties were consistently explained in terms of stronger equatorial ligand field of the heme iron in a narrower coordination cavity. Similarities of azaheme to verdoheme were also pointed out.


INTRODUCTION

The heme in hemoproteins is a tetrapyrrole compound cyclized through the four meso carbons. Replacement of one or more of the bridging carbon(s) with nitrogen(s) leads to the special porphyrinoids known as azaporphyrins (Jackson, 1978). The most familiar azaporphyrin is the tetraaza compound, phthalocyanine (Hoffman and Ibers, 1983; Lever et al., 1993).

Monoazaporphyrin (Jackson, 1978), a formal 1:3 hybrid of phthalocyanine and porphyrin, has received very limited attention as compared with the parent macrocycles. Fischer and Friedrich(1936) first synthesized monoazaporphyrin in extremely low yield. The synthesis has been much improved by Harris et al.(1966) and Engel et al. (1978). The efficient and general synthesis is essential because it first allows us to prepare sufficient quantity of the monoazaporphyrin bearing a molecular architecture like native porphyrins. Attachment of the two propionyl residues at the 6th and 7th sites of heme periphery, although not always essential (Neya et al., 1988, 1993), suggests steady incorporation of the synthetic azaporphyrin into the protein matrix.

Nitrogen atom has a smaller van der Waals radius and is more electronegative than carbon atom. Both conformational distortion at meso nitrogen and electron withdrawing capacity of the nitrogen should coincidentally affect the reactivity of the chelating metal ions, as has been extensively examined in phthalocyanines (Jackson, 1978; Thompson et al., 1993). Thus, it is likely that the reactivities of monoazahemin-bound protein toward the ligands are affected to a considerable extent. We employ in the present work -azamesoporphyrin XIII (see inset of Fig. 1), a new compound derived through the established synthetic route (Engel et al., 1978), as the prosthetic group of Mb.() The iron complex, as we demonstrate below, forms stable complex with apoMb. In consequence, one may inquire about the biological activity and spectral properties of the monoazahemin in Mb under various ligation and oxidation states. The Mb could contribute to the new biological chemistry of Mb as well as the development of the coordination chemistry of azahemins.


Figure 1: Electronic spectrum of purified ferric azaheme Mb in 0.1 M Tris buffer at pH 7.0 and 20 °C. The inset denotes the structure of the prosthetic group.




MATERIALS AND METHODS

-Azamesoporphyrin XIII and the Iron Complex

1,19-Dideoxy-3,17-diethyl-8,12-di(methoxycarbonylethyl)-2,7,13,18-tetramethyl-1,19-dibromobiladiene-ac dihydrobromide (100 mg), prepared from 5-bromo-4-ethyl-3-methyl-2-formylpyrrole (Siedel and Fischer, 1933) and 3,3`-di(methoxycarbonylethyl)-4,4`-dimethylpyrromethane-5,5`-dicarboxylic acid (Fuhrhop and Smith, 1975), was refluxed in 20 ml of methanol for 16 h in the presence of 500 mg of sodium azide (Engel et al., 1978). The resultant crude -azamesoporphyrin XIII, after evaporation of the solvent, was purified with silica gel column using 2% methanol in dichloromethane as eluent. Slow evaporation of the concentrated solution from methanol-dichloromethane mixture (1:1, v/v) under low heating afforded the title azaporphyrin as deep violet micro-needles (20 mg, 39%). CHNO requires C, 70.57; H, 6.94; N, 11.74: Found C 70.57; H, 7.04; N, 11.45. m/e 595 (M) for 595.7. -2.73 (2H, br s, NH), 1.81 (6H, t, CHCH), 3.22 (4H, t, CHCHCO), 3.53 and 3.54 (each 6H, s, ring CH), 3.64 (6H, s, OCH), 3.96 (4H, q, CHCH), 4.32 (4H, t, CHCHCO), 9.85 (2H, s, meso H) and 10.04 (1H, s, meso H). () in chloroform; 376 nm (95 mM cm), 503 (6.5), 534 (18.8), 559 (6.7), and 610 (20.0).

The iron complex was prepared by refluxing the azaporphyrin with FeClnHO in dimethylformamide under nitrogen atmosphere (Adler et al., 1970), oxidized with air, and purified on a silica gel column with chloroform-methanol mixture (9:1, v/v). () in chloroform: 363 nm (85 mM cm), 457 (8.7), 486 (8.8), 552 (11.9), 630 (3.6), and 660 (3.5). The monoazahemin ester was hydrolyzed in a refluxing methanol-water mixture containing 1% KOH as described by Fuhrhop and Smith(1975) for ordinary porphyrin diesters.

Reconstituted Mb

Sperm whale Mb was available from Sigma (type II). ApoMb (Teale, 1959), mixed with a 1.2-equivalent amount of the azahemin, was dialyzed against cold 10 mM bis-Tris buffer, pH 6.0 (Asakura, 1978). The solution was applied on a short CM-cellulose (Whatman, CM-52) column previously equilibrated with 10 mM Tris buffer, pH 7.0. The purified Mb was eluted from the column with a linear gradient of Tris buffer, 10-100 mM, at pH 7.0 and 4 °C. Fractions with an absorbance ratio A/A > 3.3 were collected and combined. All of the above manipulations smoothly proceeded to afford a reconstitution yield of 80-90%. The protein solution was concentrated for proton NMR measurements with an Amicon ultrafiltration apparatus equipped with PM-10 membrane. Concentration of the eluted Mb was determined with 105 mM cm at 385 nm on the basis of the pyridine hemochromogen spectrum with 73.2 mM cm at 394 nm and 48.0 mM cm at 553 nm in 0.1 M NaOH-pyridine mixture (1:1, v/v).

Spectroscopy

Optical absorption measurements were carried out on a Shimadzu MPS-2000 instrument equipped with thermostatted sample holder. Proton NMR spectra were recorded at 300 MHz with a Varian XL-300 spectrometer. EPR spectra were obtained at X-band (9.35 GHz) microwave frequency by using a Varian cavity with a home-built EPR spectrometer with 100 kHz field modulation (0.5 mT). An immersion double Dewar flask was used for liquid-helium temperature EPR measurements.

Ligand Binding

Ligand binding experiments were achieved with the visible absorption spectrometer. An oxygen equilibrium curve was recorded on an automatic recording apparatus (Imai, 1981) in the presence of an enzymic reducing system (Hayashi et al., 1973). The CO association constant k was measured by a UNISOK flash-photolysis apparatus as described previously (Neya et al., 1994). The dissociation constant k for CO was determined by the CO-NO replacement method. The association constant of O was obtained by the stopped-flow measurements. The k for O was estimated by dividing k with the equilibrium constant. Attempted flash photolysis for the NO Mb was unsuccessful because the derivative was not photodissociable. Rapid NO binding could not be monitored with the stopped-flow apparatus. The slow cyanide binding to deoxy Mb was monitored with a Shimadzu MPS-2000 spectrophotometer.

Autoxidation Rates

The ferric azaheme Mb was reduced with 3-fold molar excess of sodium dithionite powder, loaded onto and eluted from a Sephadex G-25 (Pharmacia Biotechnology Inc.) column with air-saturated 0.1 M Tris, pH 7.0, in a cold room. The oxy Mb, diluted with the same buffer, was used immediately for the autoxidation measurements at 30 °C on the spectrophotometer. Absorbance of the Soret peak was monitored. The end point was determined after complete oxidation of the oxy Mb by adding excess potassium ferricyanide. The oxidation of mesoheme-substituted oxy Mb was similarly monitored. The results were analyzed with an equation d(MbO)/dt = -k(MbO), where k is the apparent autoxidation rate.


RESULTS

Prosthetic Group

We first determined the pK (Hambright, 1975) of -azamesoporphyrin XIII to quantitatively evaluate the influence of electronegative meso nitrogen. With lowering pH of the porphyrin solution from 5 to 1 in 2.5% aqueous sodium laurylsulfate at 20 °C, the Soret absorption in a 340-460 nm region reversibly changed with isosbestic points at 377, 388, and 426 nm (results not shown). The optical shifts were ascribed to the third protonation at the pyrrole nitrogen, rather than at meso nitrogen, because the meso nitrogen is protonated only at high sulfuric acid concentrations (Grigg et al., 1973; Jackson, 1978). The analysis of the 398-nm transition with a single-proton equilibrium afforded a pK of 3.0 ± 0.15. Thus the introduction of a single nitrogen atom at the meso bridge sufficiently decreases pK = 5.8 of mesoporphyrin (Hambright, 1975) like the strongly electron-withdrawing formyl groups in the diformyl porphyrin (Sono et al., 1976).

Reconstituted Mb

The spectrophotometric titration of the ferric azahemin to apoMb confirmed a 1:1 binding between the two components. In the optical spectrum of the purified Mb in Fig. 1, the absorbance ratio A/A = 3.5 was reproducibly constant for several preparations. The entire spectral profile with main bands at 455, 495, 540, and 637 nm looks like that of catalase (Schonbaum and Chance, 1976). The decreased absorbance ratio A/A = 3.5, as compared with 5.0 of native Mb (Rothgeb and Gurd, 1978), arises from inherently lower Soret absorption of the prosthetic group containing a meso nitrogen (Jackson, 1978).

We recorded the pH-dependent optical spectra to deduce the coordination environment. The 385-nm absorption decrease in Fig. 2can be fitted with a single-proton equilibrium of pK = 8.5. The result suggests the presence of an iron-bound water molecule, which dissociates into hydroxide with increasing pH, as in many other aquomet Mbs (Tamura et al., 1973a; Sono and Asakura, 1976). We examined the proton NMR spectrum to further deduce the coordination state in the deoxy Mb. The NMR spectrum in Fig. 3agrees with those of the native (La Mar et al., 1977) and etioheme-substituted Mbs (Neya et al., 1989). The exchangeable 85.2 ppm signal is unambiguously assigned to the iron-bound proximal histidine because such a large shift can be induced solely by the direct iron-spin transfer through the iron-histidine bond (La Mar et al., 1977). Appearance of the single peak provides evidence for the selective interaction between the azahemin and apoMb, the formation of the iron-histidine bond, and 5-coordinate high spin state of the ferrous heme iron.


Figure 2: Acid-alkaline transition of ferric azaheme Mb (9 µM) in 5 mM Tris at 20 °C. A, the 385-nm peak decreases with increasing the pH from 7.03 to 7.19, 7.34, 7.54, 7.73, 7.93, 8.13, 8.28, 8.43, 8.56, 8.83, 9.08, 9.47, 9.81, and 10.91. B, analysis of the 385-nm transition with a pK = 8.51 ± 0.32 and a slope = 0.98 ± 0.03.




Figure 3: Proton NMR spectrum of deoxy azaheme Mb. The spectrum was recorded in HO-DO (9:1, v/v) mixture containing 3 mM Mb and 0.1 M Tris at pH 7.0 and 20 °C. The 85.2 ppm resonance is an exchangeable NH proton from the proximal histidine. DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate.



EPR is particularly sensitive to the spin and coordination states of Mb. Our initial measurement for metMb at 80 K was unsuccessful to afford no signals. The signal with g = 5.3 (part A of Fig. 4 ), appeared only at liquid helium temperature, is similar to that of an intermediate spin protein, Aplysia metMb (Hori et al., 1990), and quite distinct from the typical ferric high spin spectrum of native aquomet Mb. Addition of sodium fluoride or sodium azide to azaheme Mb produced the typical high and low spin spectra as illustrated. The EPR spectrum of the ferrous NO complex was anomalous (part B of Fig. 4). Contrary to native Mb, the fleshly prepared azaheme Mb exhibits a typical 5-coordinate type spectrum with g = 2.098 and g = 2.010 and a hyperfine coupling constant A = 2.3 mT, similar to that found in the hemoglobin bound with inositol hexaphosphate (Maxwell and Caughey, 1976). The spectrum gradually replaced with that of 6-coordinate nitrosyl heme. On standing the sample in ice bath for >10 h, only the signal from 6-coordinate nitrosyl Mb with A = 3.1 mT remained.


Figure 4: EPR spectra of azaheme Mb derivatives. A, ferric complexes at 5 K. The topspectrum is for native aquomet Mb. B, ferrous NO complexes at 80 K. A 0.8 mM Mb in 0.1 M Tris buffer at pH 7.0 was used for both observations.



Ligand Binding and Absorption Spectra

Ferric azaheme Mb binds a variety of exogenous ligands. With addition of sodium azide, a new Soret peak appeared at 398 nm with well-defined isosbestic points at 394 and 448 nm. Analysis of the intensity decrease at 385 nm resulted in an affinity of 1440 M. Optical titration confirmed the cyanide and fluoride binding to the Mb as well with 140,000 M and 3.3 M, respectively.

The ferric protein is easily reduced with sodium dithionite or enzymic reducing system to form stable ferrous Mb that reversibly binds O, CO, and NO. The analysis using the Hill plot in Fig. 5revealed an unprecedentedly high O affinity of P = 0.01 mm Hg. This is indeed 40-100-fold higher than those reported for mesoheme-substituted or native Mbs (Sono and Asakura, 1975). The oxy azaheme Mb was fairly stable; the autoxidation rate was 0.34 10 min at 30 °C while we obtained 5.2 10 min for oxy mesoheme Mb under the same conditions (results not shown). The CO binding profiles of azaheme Mb were comparable with those of native Mb. Tables I and II summarize the ligand-binding results.


Figure 5: A Hill plot of oxygen equilibrium for ferrous azaheme Mb. P, partial oxygen pressure in mm Hg; Y, fractional saturation with oxygen. The measurement were made in 0.1 M phosphate buffer at pH 7.0 and 20 °C in the presence of enzymic reducing system. P at half saturation is 0.01 mm Hg and the Hill coefficient n is 0.97.



The electronic spectra of the ferric and ferrous derivatives are presented in Fig. 6and I. The visible bands are unusually prominent relative to the Soret region, being about 10-60% as intense. The Soret peaks are broader and move toward blue by 10-30 nm than those of mesoheme Mb (Tamura et al., 1973a, 1973b). The CO complex exhibits two split Soret bands, in contrast to a single prominent peak of native Mb (Rothgeb and Gurd, 1978).


Figure 6: Visible spectra of representative azaheme Mb derivatives. A, ferric complexes (CN, - - - -; F, -; OH, ). B, ferrous complexes (deoxy, - - - -; O, -; CO, ). The spectra were recorded in 0.1 M Tris buffer at pH 7.0 and 20 °C in the presence of saturating amounts of the ligands.



Azaheme Mb exhibits novel and unique ligand-binding behaviors. We found that cyanide, pyridine, and imidazole bind to the ferrous Mb. Although Keilin and Hartree(1955) reported weak cyanide binding to native deoxy Mb at pH 9.3, the affinity at neutral pH is extremely low so that the cyano complex is formed only transiently upon reduction of cyanmet Mb (Olivas et al., 1977; Bellelli et al., 1990). Pyridine and imidazole have never been appreciated as the ligands for native ferrous Mb. On the other hand, the cyanide, pyridine, and imidazole compounds of azaheme Mb were fairly stable. The imidazole and pyridine derivatives were reddish-violet and the cyano Mb was bright blue-green in solution. Fig. 7shows the imidazole titration to the deoxy Mb examined under nitrogen atmosphere. The affinities for pyridine, imidazole, and cyanide were determined to be 350 M, 7.1 M, and 440 M, respectively. We obtained a k = 0.70 M s for cyanide at pH 7 and 20 °C. No EPR signals were detectable for these three derivatives, suggesting their ferrous low spin configuration. The visible spectra and binding constants of the new complexes are summarized in Fig. 8and . Azide, thiocyanate, and fluoride did not bind to the deoxy Mb.


Figure 7: Imidazole binding to deoxy azaheme Mb. A, imidazole concentration increases from 0 to 9.30, 19.4, 36.2, 54.4, 75.4, 97.9, and 124 mM in 0.1 M Tris at pH 7.0 and 20 °C. B, analysis of the ligand binding equilibria monitored at the wavelengths where the largest changes were observed.




Figure 8: Visible spectra of the ferrous complexes of azaheme Mb. These were recorded in 0.1 M Tris buffer at pH 7.0 and 20 °C in the presence of high concentrations (0.5 M KCN, 0.3 M pyridine, and 3 M imidazole) of the ligands.




DISCUSSION

Mb with Azahemin

The 1:1 binding stoichiometry of the azahemin with apoMb and the formation of the iron-histidine bond provide solid experimental support for the normal Mb reconstitution. The apparently normal binding of oxygen and other ligands is consistent with regular azaheme insertion. It is very likely that the entire globin fold and the side chain conformations around the prosthetic group are least perturbed after insertion of azamesohemin, as in the hemoproteins reconstituted with mesohemin (Padlan, 1975; Seybert and Moffat, 1977) or even with iron porphine (Neya et al., 1993). Smooth preparation of the reconstituted Mb may be understood from the structural similarity of the azaheme bearing two propionyl side chains with natural hemes.

Contracted Iron Cavity

The EPR signal at g = 5.2 (Fig. 4) indicates dominant intermediate spin (S = 3/2) character (Weber, 1982; Maltempo et al., 1979) for azaheme-substituted aquomet Mb, in contrast with native aquomet Mb with high spin (S = 5/2) heme iron. The anomalous spin state of azaheme Mb primarily comes from insertion of a meso nitrogen.

The C-N(meso) bond in chloro Fe(III) phthalocyanines, 1.317-1.320 Å (Palmer et al., 1985; Fitzgerald et al., 1992) is shorter than the C-C(meso) bond in corresponding porphyrins, 1.378-1.401 Å (Koenig, 1965; Scheidt and Finnegan, 1989) due to a smaller atomic radius of nitrogen atom. The bond angle at the meso nitrogen, 121° in tetraazaporphyrin (Palmer et al., 1985; Fitzgerald et al., 1992), is slightly smaller than 124-126° at meso carbon in porphyrin (Koenig, 1965; Scheidt and Finnegan, 1989). Combination of these two factors results in a smaller central cavity and stronger in-plane ligand field of the phthalocyanine iron atom. According to the recent x-ray result, the structural features found in the tetraazaporphyrins are moderately conserved in Fe(III) monoazaporphyrin (Balch et al., 1993b). The average Fe(III)-N(pyrrole) distance, 2.044 Å in monoazahemin (Balch et al., 1993b), is smaller than 2.055-2.062 Å in ordinary hemins (Koenig, 1965; Scheidt and Finnegan, 1989), so that the hole size of monoazahemin is somewhat more constricted. Consequently, an intermediate (S = 3/2) spin character found in the aquomet derivative of azaheme Mb directly reflects a narrower coordination hole, as found in some other hemoproteins (Maltempo et al., 1979; Weber, 1982).

Another support for a narrower metallo cavity in azaheme Mb comes from the acid-alkaline transition (Fig. 2). The pK= 8.5 is about one pK unit larger than 7.3 for 2,4-diformylhemin Mb (Sono and Asakura, 1976), despite an identical pK of 3.0 for the two free-base porphyrins. The larger pK of azaheme Mb suggests stronger H-OH bond of the coordinating water molecule and hence weaker Fe(III)-HO interactions. The weaker axial ligand field in tazaheme Mb is consistent with the stronger Fe(III)-N(pyrrole) interactions in the contracted central cavity of azaheme.

Additional evidence arises from greater reluctance of the oxy Mb to autoxidation. Consistent with the high equilibrium affinity and much slower dissociation rate for O (Springer et al., 1989; Brantley et al., 1993), azaheme Mb is 15-fold more stable than mesoheme Mb. The stability of oxy azaheme Mb may due to enhanced back bonding from Fe(II) into orbitals of azaporphyrin, as has been pronounced in the tetraazaporphyrin case (Ercolani et al., 1983; Fitzgerald et al., 1992). The enhanced back bonding from Fe(II) reflects contraction of the iron hole.

Meso Nitrogen and Ligand Binding

It is well known that phthalocyanine with a smaller cavity is a better -donor than porphyrin (Kennedy et al., 1986; Fitzgerald et al., 1993). Inherent phthalocyanine character found in the monoazaheme in Mb, as pointed out in the preceeding discussion, rationally suggests increased ability of the pyrrole as electron donor. This could induce repulsive interactions between iron d

The altered ligand-binding behaviors in ferrous azaheme Mb (Tables I and II) may be similarly explained. Narrower cavity causes greater Fe(II)-to-macrocycle back bonding and increases the effective positive charge on the Fe(II). The x-ray results of tetraazaporphyrin supports this interpretation. The Fe(II)-N(imidazole) bond length in phthalocyanine (1.946 Å; Ercolani et al., 1991) is appreciably shorter than in porphyrin (2.014 Å; Scheidt and Gouterman, 1983). Consistently, ferrous tetraazaheme (Fitzgerald et al., 1992) has much larger affinities for pyridine and imidazole than ferrous porphyrin (Brault and Rougee, 1974). It is also notable that CN dissociates from Fe(II) with 0.002 s for azaheme Mb while 0.14 s is reported for native Mb (Bellelli et al., 1990) under comparable conditions. A larger iron-to-azaheme back bonding accounts for increased stability of the Fe(II)-ligand complexes of azaheme Mb.

As shows, O is much more susceptible to the narrower iron cavity of azaheme than CO. The results may be rationalized by the difference in the and bonding properties of the two ligands. It is genarally accepted that CO is a better acceptor and poorer donor than O (Shriver et al., 1994). The altered discrimination between CO and O in azaheme Mb suggests weakened bonding and/or enhanced interaction in the Fe(II)-ligand bonds. It is also notable that the NO azaheme Mb initially forms a 5-coordinate complex (Fig. 4). We propose that the Fe(II)-NO bond in azaheme Mb is much stronger than that in native 6-coordinate Mb. The NO azaheme Mb correspondingly is not photodissociable. Since NO is a better -acceptor than CO, the cleavage of the Fe(II)-histidine bond in azaheme Mb indicates enhanced -bonding character of the Fe(II)-NO interactions.

Similarity with Verdoheme

In the liver and spleen of many animals, protoheme IX is degraded into biliverdin by heme oxygenase. In the degradation process, an oxygen atom is inserted to the meso bridge to produce an intermediate compound called -oxoprotoheme or verdoheme (Saito and Itano, 1986; Balch et al., 1993a). Replacement of the porphyrin meso bridge by an oxygen atom infers close similarity of oxoheme with azaheme because both elements are more electronegative and smaller in atomic radii than carbon atom. Indeed, azaheme (Balch et al., 1993b) has a narrower metallo cavity and forms the bis-halogen adduct in the ferric state as oxohemin does (Balch et al., 1993a). It is notable that ferrous azaheme Mb binds cyanide like the ferrous Mb containing verdoheme (Fujii, 1990). The visible spectrum of ferrous cyano azaheme Mb is a typical verdoheme-type spectrum (Saito and Itano, 1986; Fujii 1990) to support spectroscopic similarity between azaheme and verdoheme. In ferrous azaheme Mb, a 60 nm blue-shift of the visible band is observed upon the ligand replacement from cyanide to CO, in good agreement with the results that the CO and cyanide adducts of verdohemochrome in Mb show visible bands at 655 and 707 nm, respectively (Fujii, 1990). The verdoheme in heme oxygenase is not easy to characterize due to the spontaneous degradation into biliverdin. Oxoheme and azaheme are chemically similar to each other except for the enhanced stability of the latter against reducing agents and molecular oxygen. The possibility exists that azaheme is a stable substitute for labile verdoheme in heme oxygenase. A neutral imidazole has been proposed as the iron-bound ligand in heme oxygenase (Takahashi et al., 1994a, 1994b; Sun et al., 1993) as in Mb. This observation suggests that azaheme Mb itself serves as a close model to investigate unexplored physicochemical properties of the heme oxygenase substituted with verdoheme.

  
Table: Comparison of the ligand affinities in M between azaheme and native Mbs in 0.1 M Tris at pH 7.0 and 20 °C


  
Table: Rate and equilibrium (accurate to ±10%, at pH 7.0 and 20 °C) for O and CO binding to the ferrous Mbs containing the porphyrins with pK = 3.0


  
Table: Visible absorption parameters of the Mb containing -azamesohemin XIII



FOOTNOTES

*
This work was supported by a grant-in-aid from the Scientific Research Foundation of Kyoto Pharmaceutical University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan. Tel.: +81-75-595-4664; Fax: +81-75-595-4762.

The abbreviations used are: Mb, myoglobin; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.


ACKNOWLEDGEMENTS

We are much indebted to Drs. Tetsuhiko Yoshimura and Hiroshi Fujii (Yamagata Research Institute of Technology) for helpful discussions. We also thank anonymous reviewers for valuable comments on the original manuscript.


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