How does heme axial ligand deletion affect the structure and the function of cytochrome b562?

Noriho Kamiya1, Yuko Okimoto1, Zhen Ding1, Hiroko Ohtomo1, Masafumi Shimizu1, Atsushi Kitayama1, Hisayuki Morii2 and Teruyuki Nagamune1,3

1 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku,Tokyo 113-8656, Japan and 2 National Institute of Bioscience and Human-Technology NIBH-E6, Tsukuba, Ibaraki 305-8566, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
We have recently generated a new mutant of cytochrome b562 (cytb562) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb562). The M7A cytb562 can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb562, thermal transition analyses of M7A cytb562 were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb562 is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb562 was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb562, whereas the functional conversion of cytb562 is successfully achieved by the loose folding together with the open coordination site.

Keywords: folding stability/four-helix bundle protein/functional conversion/heme binding/oxidative reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
Hemoproteins show diverse functions such as electron transport, oxygen storage and delivery, and catalysis by effectively altering either the coordination of the heme iron or the orientation of amino-acid residues around the prosthetic group toward each function. Since many of the various hemoproteins with different functions carry the same prosthetic group such as iron-protoporphyrin IX, functional conversion between hemoproteins by protein engineering has been a major challenge. In fact, owing to their structural alignment, the conversion of myoglobin to catalytic heme enzyme has been extensively studied by site-directed mutagenesis. With the alteration of amino acid residues especially in the distal side of heme, sperm whale and horse heart myoglobins were found to be peroxygenase (Ozaki et al., 1996Go; Matsui et al., 1999Go) and peroxidase (Wan et al., 1998Go), respectively.

A cytochrome, whose original function is electron transfer, has also been a promising candidate for the objective. The generation of demethylase activity with bis-histidine ligated cytochrome b5 (cytb5) was demonstrated by replacing one of the heme axial ligands, His39, by Met (Sligar et al., 1987Go). The substitution of the axial Met80 by Ala of horse cytochrome c yielded a novel semisynthetic cytochrome that exhibits globin-like function (Bren and Gray, 1993Go). Recently, there have been several reports on the formation of verdoheme-containing mutant proteins of cytb5 (Rodríguez and Rivera, 1998Go) and cytb562 (Rice et al., 1999Go) by altering the axial His to Met. These observations imply that increasing the accessibility of exogenous ligands to the heme iron is crucial for the generation of new function (i.e. functional conversion). However, the deletion of one of the axial heme ligands often led to the loss of heme-binding ability (Nikkila et al., 1991Go; Ihara et al.2000Go).

Escherichia coli cytb562 is a four-helix bundle protein containing a non-covalently bound iron-protoporphyrin IX. The two axial heme ligands are Met7 on the first helix and His102 on the fourth helix (Itagaki and Hager, 1966Go; Xavier et al., 1978Go; Mathews et al., 1979Go). Since the solution structures of the apo- and holo-forms of cytb562 were available (Feng et al.1994Go; Arnesano et al., 1999Go), it has been a suitable model hemoprotein for structural characterization (Feng and Sligar, 1991Go; Robinson and Sligar, 1993Go; Robinson et al., 1997Go,1998Go). The abundunt information together with the high-level expression system in E.coli (Nikkila et al., 1991Go) makes cytb562 an ideal model hemoprotein for changing the functional properties by protein engineering (Barker et al.1996Go; Barker and Freund, 1996Go; Springs et al., 2000Go). However, the study on the axial ligand mutation of cytb562 is still limited (Barker et al.1996Go; Barker and Freund, 1996Go). To our knowledge, no study has been performed on the deletion of one of the heme axial ligands probably due to the lack of the capability of heme-binding upon such mutation (Nikkila et al., 1991Go).

Recently, we have prepared a new mutant of cytb562 in which Met7 is replaced by Ala (M7A cytb562). It was expressed as a holoprotein in the periplasm of host E.coli cells, as well as a carbonmonoxy form (Kamiya et al., in preparation ). This implies that an exogenous ligand or a substrate can bind to the heme iron, indicating the generation of new enzymatic function. Furthermore, M7A cytb562 gives us the opportunity to evaluate the role of Met7 ligation to the heme iron on the integrity of the four-helix bundle structure of cytb562. In this study, we investigated the structural and functional properties of M7A cytb562 in detail. This mutant is the first example showing the successful generation of an open pocket for substrate binding within cytb562 scaffold.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
Sample preparation

The preparation of M7A cytb562 is described elsewhere in detail (Kamiya et al., in preparation ). Since the mutant protein was expressed as a carbonmonoxy form, the apoprotein of M7A cytb562 (apoM7A cytb562) was prepared at first (Teale, 1959Go). Then it was successfully reconstituted with exogenous hemin to obtain the holo form (holoM7A cytb562) owing to the fairly high affinity of hemin to the apo form (Kd = 87 ± 17 nM; Kamiya et al., in preparation ). The protein concentration was determined by amino acid analysis. The millimolar extinction coefficients ({varepsilon}) of holoM7A cytb562 and its derivatives were determined by the pyridine-hemochromogen method (Fuhrhop and Smith, 1975Go). The absorbance at 418 ({varepsilon} = 117.4 mM–1 cm–1) and 277 nm ({varepsilon} = 3.0 mM–1 cm–1) were used for the calculation of the concentrations of oxidized wild-type cytb562 (WT cytb562) and the apo form (apoWT cytb562), respectively (Itagaki and Hager, 1966Go; Feng and Sligar, 1991Go).

Spectroscopic measurements

UV-visible absorption spectra of holoM7A cytb562 and the samples with exogenous ligands were measured on a Ubest 550 spectrophotometer (Jasco, Japan) equipped with a thermostated cell holder. Far-UV circular dichroism (CD) spectra were measured on a JASCO J-630 CD spectropolarimeter (Jasco, Japan). The measurements were performed with a protein concentration of 30 µM in a quarts cell with 0.02-cm path length. The CD spectra were recorded with an increment of 0.2 nm, a scan rate of 20 nm min–1, a response time of 1 s and sensitivity of 20 mdeg. No data smoothing was conducted.

Thermal transition analysis

Thermal transition of apoM7A and holoM7A cytb562s was measured by simultaneously monitoring the temperature and the molar ellipticity at 222 nm according to the procedure previously described (Morii et al., 1999Go). In the case of holoM7A cytb562, the measurement of Soret absorbance at 407 nm was also performed concurrently. The measurement was performed with a protein concentration of 30 µM in a quart cell with 0.1-cm path length. The heating rate was 0.75 K min–1. The thermal transition curves obtained were fitted with the weighted-mean combination of the two linear functions for the folded and unfolded states to determine Tm and {triangleup}Hv values directly (Morii et al., 1999Go). The free energy of stabilization ({triangleup}G) was calculated by the Gibbs–Helmholtz equation

Proteolysis experiments

ApoM7A, holoM7A and WT cytb562s were employed for proteolysis experiments. All reactions were conducted with 0.5 mg ml–1 protein solution containing 0.1 mg ml–1 trypsin in 10 mM potassium phosphate buffer (pH 7.0 or 8.0) at 15°C. The aliquots of the reaction mixtures were derived at 5 and 24 h after addition of trypsin. They were immediately subjected to the sample preparation for SDS–PAGE (12.5% PAG) (Laemmli, 1970Go).

Assay of peroxidase activity and steady-state kinetics of oxidation reaction

Peroxidase activity was evaluated by measuring the initial oxidation rate of o-methoxyphenol (guaiacol, Tokyo Chemical Industry, Japan) or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma Co., USA) with hydrogen peroxide (Wako Pure Chemical Industries, Japan). Hemin (Tokyo Chemical Industry, Japan) and horse heart myoglobin (Mb, minimum 90%, salt-free, lyophilized sample, Sigma Co., USA) were used as received. The product formation was detected by the change of absorbance at 470 nm (guaiacol, {varepsilon} = 26 000 M–1 cm–1, Ozaki and Ortiz de Montellano, 1995) or 730 nm (ABTS, {varepsilon} = 14 000 M–1 cm–1, Matsui et al., 1999). The concentration of hydrogen peroxide was determined as previously described (Cotton and Dunford, 1973Go). Steady-state kinetics were conducted with 1 µM holoM7A cytb562 or Mb in 10 mM potassium phosphate buffer (pH 7.0) at 15°C. The concentration of hydrogen peroxide was 1 mM and the concentration of substrates was varied (1.1–110 µM guaiacol with holoM7A cytb562, 0.25–10 mM guaiacol with Mb; 10–200 µM ABTS with M7A cytb562, 0.2–3 mM ABTS with Mb ). The initial reaction rates obtained were fitted to the Michaelis–Menten equation by the non-linear least-squares method with the software KaleidaGraph on a personal computer.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
UV-visible absorption and far-UV CD spectroscopies

The UV-visible absorption spectrum of holoM7A cytb562 is shown in Figure 1Go. It shows a sharp Soret band at 407 nm ({varepsilon} = 153 mM–1 cm–1) and two visible bands at 500.5 and 632 nm ({varepsilon} = 12.1 and 6.0 mM–1 cm–1, respectively ). The spectrum is typical of a high-spin ferric heme and is completely distinct from that of WT cytb562 (Itagaki and Hager, 1966Go). The spectral change upon incorporation of an exogenous ligand (1 mM) is also shown in Figure 1Go. The UV-visible absorption spectra of cyano- and azide-complexes of holoM7A cytb562 (Figure 1Go, broken and dotted lines, respectively ) were similar to those of myoglobin (Tamura et al., 1973Go). The cyano-complex of holoM7A cytb562 exhibits absorption maxima at 422 and ~540 nm ({varepsilon} = 122 and 13.8 mM–1 cm–1, respectively ) and those of azide complex were found at 422, 543 and 575 nm ({varepsilon} = 129, 14.1 and 11.7 mM–1 cm–1, respectively ). Figure 2Go shows the far-UV CD spectra of apoM7A and holoM7A cytb562s. HoloM7A cytb562 appeared to have slightly higher {alpha}-helicity compared to the apoprotein, indicating the secondary structural change upon heme binding.



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Fig. 1. UV-visible absorption spectra of M7A cytb562 (solid line) and the cyano- (broken line) and azide- (dotted line) complexes. All spectra were measured in 10 mM potassium phosphate buffer at pH 7.0 containing 3.4 µM protein at 15°C. The concentration of sodium cyanide and sodium azide was 1 mM.

 


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Fig. 2. Far-UV CD spectra of holoM7A cytb562 (solid line) and the apoprotein (dotted line) in 10 mM potassium phosphate buffer at pH 7.0 and 10°C.

 
Stability against proteolysis

To obtain some information on the solution structure and the stability of M7A cytb562, the proteolysis was investigated. The digestion of apoM7A, holoM7A and WT cytb562s with trypsin at 15°C and pH 7.0 (Figure 3aGo) or 8.0 (Figure 3bGo) was monitored by SDS–PAGE. As shown in the figure, WT cytb562 was completely resistant to proteolysis even after incubation for 24 h at both pH values. On the other hand, apoM7A cytb562 was readily digested in 5 h independent of pH. ApoWT cytb562 gave the same results as apoM7A cytb562 (data not shown). On the other hand, holoM7A cytb562 was gradually subjected to proteolysis at pH 7.0. At pH 8.0, the optimal pH for tryptic activity, a significant portion of holoM7A cytb562 was digested in a 5 h treatment. These results imply that the folding stability of holoM7A cytb562 is much lower than WT cytb562, although the heme binding to apoM7A cytb562 slightly increases the stability against proteolysis.



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Fig. 3. Time courses of proteolyses of apoM7A, holoM7A and WT cytb562s in 10 mM potassium phosphate buffer at pH (a) 7.0 and (b) 8.0 monitored by SDS–PAGE [left-end lane in (a), molecular marker; lanes 1, 4 and 7, intact proteins; lanes 2, 5 and 8, after 5 h digestion; lanes 3, 6 and 9, after 24 h digestion).

 
Thermal transition analysis

Thermal transition analyses were conducted to obtain key parameters of the thermodynamic characteristics of M7A cytb562. Since the UV-visible spectral change of holoM7A cytb562 showed a clear isosbestic point upon thermal unfolding (data not shown), we assumed the simple two-state thermal transition between native and thermally unfolded forms as well as the case of WT cytb562 (Feng and Sligar, 1991Go; Robinson et al., 1998Go). Figure 4Go shows the thermal transition profiles obtained. The unfolding curves fitted well to the assumption by the method previously described (Morii et al., 1999Go). The analysis of apoM7A cytb562 yields a Tm of 48.4°C and {triangleup}Hv of 44.7 kcal mol–1. On the other hand, the Tm and {triangleup}Hv values for holoM7A cytb562 on the basis of CD measurement were determined to be 52.2°C and 47.0 kcal mol–1, respectively. Therefore, it appeared that the binding of heme to apoM7A cytb562 increased the Tm and {triangleup}Hv values by about 4 K and 2 kcal mol–1, respectively. However, significant destabilization in the folding of cytb562 was evident by the replacement of Met7. The {triangleup}Hv and Tm values of WT cytb562 were reported to be 104.2 kcal mol–1 and 66.99°C at pH 7.4, respectively (Robinson et al., 1998Go). This means that a decrease of about 60 kcal mol–1 in {triangleup}Hv and 15 K in Tm is a result of the lack of Met7 ligation to the heme iron. Moreover, the trace of the unfolding curve obtained from the Soret absorption change of holoM7A cytb562 seemed to be inconsistent with that from the CD result. However, the analysis gave the same result for Tm (52.2°C), whereas the {triangleup}Hv value was 57.2 kcal mol–1, which is a somewhat higher value compared to that from CD measurement. In the subsequent analyses, Tm and {triangleup}Hv values obtained from CD measurement were employed.



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Fig. 4. Thermal transition curves of apoM7A cytb562 (broken line) and holoM7A cytb562 (solid line) obtained from the change of molar ellipticity at 222 nm and that of holoM7A cytb562 obtained from the Soret absorption change at 407 nm (dotted line).

 
When the {triangleup}Hv values of apo- and holo-forms of M7A cytb562 and the {triangleup}Hv value of the apoWT cytb562 (47.4 kcal mol–1 and Tm of 54.04°C at pH 7.4, Robinson et al., 1998) were plotted against each Tm value, the slope of the regression line was found to be 0.49 (r2 = 0.996 ). This value is within the range of heat capacity change ({triangleup}Cp) reported for the apoWT cytb562 (0.56 ± 0.23 kcal K–1 mol–1, Robinson et al., 1998). Hence, the free energy of stabilization ({triangleup}G) of M7A cytb562 could be calculated using a {triangleup}Cp of 0.56 kcal K–1 mol–1. Given the parameters determined, {triangleup}G values of apoM7A and holoM7A cytb562s at 20°C can be estimated to be 3.2 and 3.7 kcal mol–1, respectively (Table IGo).


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Table I. Thermodynamic parameters obtained with M7A and WT cytb562s at 20°C
 
Comparison of peroxidase activity of holoM7A cytb562 with hemin and WT cytb562

Figure 5Go depicts the time courses of oxidation reaction of guaiacol with hydrogen peroxide by holoM7A cytb562, hemin and WT cytb562. The initial reaction rates of holoM7A cytb562 and hemin were 2.4 and 0.17 µM min–1, respectively. The 14-fold enhancement in the catalytic activity indicates the positive role of apoprotein in catalysis. The oxidation rate of WT cytb562 was substantially comparable to the auto-oxidation rate of guaiacol or ABTS without the protein. After the substrate conversion reached a plateau (Figure 5Go), the subsequent addition of hydrogen peroxide resulted in about one-third of the initial reaction rate. Hence, the reaction profile of holoM7A cytb562 might involve the inactivation process to some extent.



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Fig. 5. Time courses of the oxidation of guaiacol (10 mM) with hydrogen peroxide (1 mM) by holoM7A cytb562 (closed circles), hemin (triangles) and WT cytb562 (open circles) in 10 mM potassium phosphate buffer at pH 7.0 and 15°C. The concentration of proteins and hemin was 1 µM.

 
Steady-state kinetics of the oxidation of guaiacol and ABTS

We confirmed that the initial rates determined in the early phase of the reaction (typically, within 15 s) fitted well to the Michaelis–Menten equation. Therefore, we could obtain apparent kinetic parameters in the steady-state kinetics of guaiacol and ABTS oxidation reactions. For comparison, the catalytic properties of Mb, being known to show peroxidase activity (Yonetani and Schleyer, 1967Go; Wan et al., 1998Go; Matsui et al., 1999Go), were also investigated. The hemin-catalyzed reaction could not obey Michaelis–Menten-type kinetics. Table IIGo shows the apparent kinetic parameters obtained for holoM7A cytb562 and Mb. The specificity constants (kcat/Km) of holoM7A cytb562 appeared to be 81- and 34-fold higher compared to those of Mb in guaiacol and ABTS oxidations, respectively.


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Table II. Apparent kinetic parameters determined for the oxidation of guaiacol and ABTS
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
Cytb562 was originally classified as an electron transport protein, and has a low-spin ferric heme at neutral pH owing to Met7/His102 bis-ligation to the heme iron (Moore et al., 1985Go). In the case of holoM7A cytb562, the absorption spectrum can be assigned to a high-spin ferric heme and is similar to that of ferric myoglobin (Tamura et al., 1973Go). Upon the addition of up to 100 mM fluoride, no spectral change was observed (data not shown). These observations suggest the presence of a water molecule that coordinates to the heme iron as a sixth ligand. The water molecule was readily replaced by cyanide and azide ions (Figure 1Go), indicating that exogenous ligands are accessible to the heme iron. In addition, holoM7A cytb562 was found to be the most stable in a neutral pH region. The pH shift to either a lower or higher range caused absorption spectral change without isosbestic points (data not shown). It was reported that WT cytb562 is stable even if the medium pH was shifted from 3.0 to 10.5 (Itagaki and Hager, 1966Go). Therefore, it is concluded that the stable packing of helices by Met7/His102 bis-ligation to the heme iron is crucial for the high stability of WT cytb562 against pH shift.

The removal of heme from WT cytb562 caused a drastic change in the secondary and tertiary structures (Feng and Sligar, 1991Go; Feng et al., 1994Go). As shown in Figure 2Go, holoM7A cytb562 retained higher {alpha}-helicity than the apoprotein, implying the structural change on heme binding as well as WT cytb562 (Feng and Sligar, 1991Go). In the proteolysis experiment (Figure 3Go), holoM7A cytb562 showed somewhat higher stability compared to the apo form. This view is consistent with the results of thermodynamic analyses showing a slightly higher {triangleup}G value for holoM7A cytb562 (Table IGo). However, holoM7A cytb562 is much more sensitive to proteolysis than WT cytb562 despite the presence of heme. In fact, the stabilization energy of apoM7A cytb562 upon incorporation of heme ({triangleup}{triangleup}G = 0.5 kcal mol–1) is much lower than the 7.4 kcal mol–1 estimated for WT cytb562 (Table IGo). Interestingly, the thermodynamic parameters of holoM7A cytb562 showed a similar trend to those of apoM7A and apoWT cytb562s, indicating that the folding of holoM7A cytb562 is comparable to those of the apoprotein. Hence, the coordination of Met7 to the heme iron is crucial for the tight packing of the helices together with the increase of {triangleup}Hv and {triangleup}Cp values by the creation of a hydrophobic core in the heme crevice. On the basis of results obtained, the heme pocket of holoM7A cytb562 would be more exposed to solution than that of WT cytb562.

Since holoM7A cytb562 could bind exogenous ligands to the heme iron, the enzymatic function at this open coordination site was investigated in line with myoglobins and peroxidases. As shown in Figure 5Go, WT cytb562 shows no activity under the identical experimental condition. On the other hand, holoM7A cytb562 can catalyze the oxidation of either guaiacol or ABTS with hydrogen peroxide. The spectroscopic analysis of WT cytb562 demonstrated the intact bis-ligation (Moore et al., 1985Go; Wu et al., 1991Go). Therefore, Met7 is not labile enough for binding substrate molecules to the heme iron.

It appeared that higher peroxidase activity of holoM7A cytb562 compared to Mb was mainly due to the decrease of Km values in two reactions (Table IIGo). The results clearly demonstrate that the replacement of Met7 by Ala generates new substrate binding sites for guaiacol and ABTS with higher affinity than Mb. It was reported that a mutant of sperm whale myoglobin lacking distal histidine (H64L) showed no significant peroxidase activity (Matsui et al., 1999Go). This means that the distal histidine plays a key role for the efficient oxidation of substrates in Mb. However, in the case of holoM7A cytb562, histidine is not likely to be involved in the catalytic process based on the 3D structure of WT cytb562 (Arnesano et al., 1999Go). On the other hand, distal His42 defective horseradish peroxidase (H42A HRP) did not result in the complete loss of oxidation activity (Savenkova et al., 1996Go). In guaiacol oxidation, the low catalytic efficiency (kcat) of H42A HRP was compensated for by a smaller Km value compared to wild-type HRP (Table IIGo, Savenkova et al., 1996). HoloM7A cytb562 showed a similar trend in the two reactions investigated (Table IIGo). Hence, a plausible proposal for the better catalytic performance of holoM7A cytb562 than Mb is that the generation of the substrate binding site is easily accessible to reactive heme species. We still have not detected the reactive high-valent heme compound of holoM7A cytb562. Further studies will be required to clarify the catalytic mechanism of this mutant protein in peroxidase reactions.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
The results obtained here demonstrate the successful alteration of the heme environment of cytb562, in which a new coordination site is created by replacing one of the axial heme ligands, Met7 by Ala. In the present study, we could shed light on the role of Met7 for the stable folding of cytb562. Furthermore, the looser folding with an open axial site of holoM7A cytb562 uniquely qualified the generation of new enzymatic function. This study will open the way for the design of a novel, artificial b-type heme enzyme based on cytb562 four-helix bundle scaffold.


    Note added in proof
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 Note added in proof
 References
 
After we submitted this paper, a similar study concerning a cytochrome b562 mutant with an open axial site was published (Uno et al., 2001Go ). They successfully created a more stable mutant compared to M7A cytochrome b562 by altering the polar interactions between heme propionates and Glu residues around the heme crevice. In this study, we simply focused the contribution of Met7 ligation to the heme iron to the protein stability and the generation of new function. The results independently obtained here are therefore useful for designing an engineered heme protein as well as the recent report.


    Notes
 
3 To whom correspondence should be addressed. E-mail: nagamune{at}bio.t.u-tokyo.ac.jp Back


    Acknowledgments
 
We are grateful to Prof. S. G. Sligar for his kind gift of the plasmid encoding cytb562 gene. N.K. is indebted to Dr. Hiroyuki Wariishi of Kyushu University for his valuable discussion. We also thank Drs Masao Chijimatsu and Koji Takio of the Institute of Physical and Chemical Research (RIKEN) for experimental supports in amino acid analysis. This work was partially supported by the Biodesign Research Promotion Group of RIKEN, Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
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 References
 
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Received January 12, 2001; revised March 12, 2001; accepted March 19, 2001.





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