The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

Wen-Hu Wang1, Jun-Xia Lu1, Ping Yao1, Yi Xie2 and Zhong-Xian Huang1,3

1Chemical Biology Laboratory, Department of Chemistry and 2State Key Laboratory of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China

3 To whom correspondence should be addressed. e-mail: zxhuang{at}fudan.edu.cn


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b5 has been constructed by using gene random mutagenesis. Two variants of cytochrome b5, His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b5 >> His39Cys > His39Ser.

Keywords: cytochrome b5/heme axial ligand/heme-binding stability/heme coordination environment/random mutagenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Heme-containing proteins are an exquisite example of metalloprotein design strategies revealed by nature. By sharing the same prosthetic group, protoheme IX, various classes of hemoproteins perform a wide variety of biological activities, such as oxygen transport (e.g., myoglobin, hemoglobin; Antonini and Brunori, 1971Go), electron transfer [e.g., cytochrome b5 (cyt b5), cytochrome c; Lemberg and barrett, 1973Go] and biochemical reaction catalysis (e.g., cytochrome P450, chloroperoxidase; Dawson and Sono, 1987Go). The mechanisms by which protein structures regulate the intrinsic reactivity of the heme, therefore, are of both theoretical and practical interest. Although many structural variables provided by the peptide chain are quite influential, one of the most significant structural characteristics governing the functional properties of hemoproteins is the coordination environment of the heme iron, including type of axial ligands, the coordination number and coordination sphere.

A number of efforts have been directed to discover what influence the alteration of axial ligands has on the heme coordination environments and properties of hemoproteins. Highlights were mainly focused on cytochrome c (Hampsey et al., 1986Go; Raphael and Gray, 1989Go; Sorrell and Martin, 1989Go; Raphael and Gray, 1991Go; Wallace and Clark-Lewis, 1992Go; Bren and Gray, 1993aGo,b) and myoglobin (Egeberg et al., 1990Go; Adachi et al., 1991Go, 1993Go; Barrick, 1994Go; DePillis et al., 1994Go; Maurus et al., 1994Go; Qin et al., 1994Go; Lloyd et al., 1995Go). Artificial hemoproteins with new axial ligands often change their heme coordination environments and exhibit novel properties and/or reactivity. These findings are very helpful in understanding the structure–property–function relationships of hemoproteins.

In comparison with relatively plentiful work on cytochrome c and myoglobin, alteration studies on the axial ligation of cyt b5, another well-characterized model molecule of hemoproteins, have been somewhat limited. Unlike most of the other hemoproteins, the heme of cyt b5 is relatively more exposed, roughly a quarter of the heme group directly contacts the solvent (Mathews et al., 1972aGo,b; Durley and Mathews, 1996Go; Wu et al., 2000Go). In addition, the lack of any covalent link between the heme and protein peptide chain (observed in c-type cytochromes), and substantially lower hydrophobicity of the heme binding pocket in comparison with globins (Falzone et al., 1996Go; Manyusa et al., 1999Go), also lead to a weak interaction between heme and apo-protein in cyt b5. The heme-holding ability of cyt b5 depends mostly on the strong axial ligation provided by residues His39 and His63. Even in wild-type protein having intact bis-histidine coordination, a slow loss of the heme from holo-cyt b5 to apo-myoglobin is observed (Smith et al., 1991Go). As a result, most site-directed mutants at axial ligands of cyt b5 were reported previously to possess a marginal stability or even to lose the heme reconstitution ability (Beck von Bodman et al., 1986Go; Rodríguez and Rivera, 1998Go; Ihara et al., 2000Go).

At the same time, cyt b5 is one of the well-studied hemoproteins. The heme-containing functional fragments of this protein derived from restricted proteolysis (Strittmatter and Vellick, 1956Go) or from recombinant protein expression (Beck von Bodman et al., 1986Go; Funk et al., 1990Go; Rivera et al., 1992Go; Hewson et al., 1993Go) are small in size (ca. 90 amino acid residues) and have a good water solubility. Their three-dimensional structures have been determined by X-ray crystallography (Mathews et al., 1972aGo,b; Durley and Mathews, 1996Go; Wu et al., 2000Go) and NMR spectroscopy (Muskett et al., 1996Go; Banci et al., 1997Go; Arnesano et al., 1999Go; Banci et al., 2000Go). Based on the wealth of molecular information, more extensive studies focused on alteration of heme coordination environment of cyt b5 would further throw light on the key role played by the particular bis-histidine coordination in this protein. More importantly, this would help to understand the mechanism of how different axial ligation styles modulate heme properties and reactivity of various hemoproteins.

In this paper, we report a random mutagenesis of one of the heme axial ligands, His39, of lipase-solubilized bovine liver microsomal cyt b5 (Funk et al., 1990Go). Sixteen designed mutated genes have been constructed. All of them can be expressed efficiently in Escherichia coli but only apo-form proteins were produced. Thus, the intrinsic difficulty that alteration of the heme axial ligand would greatly reduce the heme binding stability in cyt b5 remains, even after the random screening in our system. Two variants of cyt b5, the His39Ser and His39Cys mutants were first purified in their apo-forms and then reconstituted with exogenous heme to obtain the holo-proteins. Interestingly, although the two mutants differ from each other by one atom (oxygen or sulfur) in the residues of position-39, they exhibit very distinct properties. While the His39Ser mutant shows different heme coordination styles in different oxidation states (His63-water ligation for ferric heme and His63-Ser39 for ferrous heme) similar to the coordination of heme in some other axial mutants of cyt b5 (Rodríguez and Rivera, 1998Go), the His39Cys mutant has the unique heme coordination environments His63-Fe(III)-Cys39 coordination for ferric heme and His63-Fe(II)-H2O only for the ferrous state. A novel coordination of the ferric heme iron by axial residue histidine and the substitutional axial residue cysteine was found for the first time in the His39Cys mutant. And this mimics the unique heme coordination of the natural CooA enzyme protein (Reynolds et al., 1998Go). It is interesting to note that the His39Cys mutant has much higher heme-binding stability than the His39Ser variant.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

DNA restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase and Klenow enzyme (large fragment of DNA polymerase I) were purchased from New England BioLabs. Plasmid pET-11c and expression host strain BL21(DE3)pLysS were purchased from Novagen. Hemin was obtained from Sigma (cat. no. H-2250). Original synthetic gene encoding the lipase-solubilized bovine liver microsomal cyt b5 cloned in pUC19 plasmid was a generous gift from Professor A.G.Mauk (Funk et al., 1990Go). All chemicals were of reagent grade.

Random mutagenesis at His39 of cytochrome b5

The designed mutagenic primer, 5'-TACGACCTGACT AAATTCCTGGAAGAG XXYCCGGGAGGCGAAGAAGT CCTGCGCGAACAGGCCGGCC-3', was synthesized by Sagong Corp., where ‘X’ means the equal mixture of A\T\G\C and ‘Y’ refers to the equal mixture of T\G\C. The absence of the nucleotide ‘A’ at the ‘Y’ position is to improve experimental efficiency considering the compatibility of amino acid triplet codes. The ‘XXY’ site corresponds to the triplet code of His39 in the wild-type cyt b5 and the random mutagenesis was introduced at this site. This oligonucleotide was first phosphorylated with T4 polynucleotide kinase, and then was cloned into the sites between the NgoMIV and BstZ17I in the wild-type cyt b5 gene in pUC19 plasmid by the ‘mutually primed synthesis’ method (Oliphant et al., 1986Go; Oliphant and Struhl, 1988Go). The mixture of mutated recombinant plasmids was used to transform E.coli strain JM83 for separation and amplification. After this, 60 single colonies were picked out for target gene screening by DNA sequencing. All correct mutant genes were sub-cloned into the NdeI/BamHI-linearized pET-11c vector separately by using the polymerase chain reaction (PCR) technique. Recombinant pET-11c plasmids, each containing unique mutant gene, were used to transform into expression host strain BL21(DE3)pLysS, separately. DNA sequencing was accomplished by using automatic DNA sequencers (model 377 or 3700, Applied Biosystems).

Protein expression and purification

Expression and purification of wild-type cyt b5 protein was performed according to the method of A.G.Mauk (Funk et al., 1990Go). Processing of the His39 mutants was carried out as described below.

A fresh single colony of E.coli strain, BL21(DE3)pLysS, containing the recombinant pET-11c plasmid was picked out to inoculate 50 ml LB medium containing ampicillin (50 µg/ml) and chloramphenicol (34 µg/ml) in a 250 ml flask. This was incubated with shaking at 37°C until OD600 was ~0.5. Every 3 ml of this starting culture was used to inoculate 400 ml of LB medium containing ampicillin (50 µg/ml) and chloramphenicol (34 µg/ml) in a 1000 ml flask. Cells were grown at 37°C until OD600 reached a value between 0.8 and 1.0. IPTG (isopropyl-ß-thiogalactoside) was added (final concentration 1.0 mM) to induce biosynthesis of the protein polypeptide, and after 4–5 h the cells were harvested by centrifugation. The cell paste was re-suspended in lysis buffer (50 mM Tris–HCl, 5 mM EDTA, 1 mM DTT, 10 mM PMSF, pH 7.5) and the cells were lysed thoroughly by sonication. Cell debris were removed by centrifugation. The supernatant solution containing apo-cyt b5 mutant was brought to 30% (NH4)2SO4 saturation, stirred for 3 h and then centrifuged again. The resulting supernatant was dialyzed exhaustively against ion-exchange buffer (20 mM sodium phosphate, 5 mM EDTA, 1 mM DTT, 10 mM PMSF, pH 8.0). The desalted solution was then loaded onto an anion-exchange column (DE52, Whatman, 3x20 cm) previously equilibrated with ion-exchange buffer and was washed thoroughly with the same buffer. The apo-protein was eluted with a NaCl gradient from 0 to 400 mM. Fractions containing target protein with purity >30% were collected and concentrated to ~10–20 ml by ultrafiltration. Protein solution was then applied to a column of Sephadex G-75 (fine class, Pharmacia, 2.8x100 cm) previously equilibrated with gel-filtration buffer I (50 mM sodium phosphate, 5 mM EDTA, 1 mM DTT, 10 mM PMSF, pH 8.0) twice and then the target protein with purity >80% was obtained. The resulting apo-protein solution was dialyzed against reconstitution buffer (100 mM sodium phosphate, 1 mM imidazole, pH 8.0) and reconstituted with a slight excess of hemin (freshly dissolved in 0.1 M NaOH solution). The reconstituted protein solution was concentrated and then applied to a column of Sephadex G-25 (medium class, Pharmacia, 2.5x20 cm) equilibrated with gel-filtration buffer II (50 mM sodium phosphate, 1 mM imidazole, pH 8.0) to remove free heme. The resulting protein solution was concentrated and passed through the G-75 column equilibrated with gel-filtration buffer II to obtain the purified holo-protein. The protein sample was lyophilized and stored at –70°C. All protein purifications were carried out at 4°C and protein purity was determined by SDS–PAGE analysis (with the gel image and analysis system, GDS 7500, UVP Company, USA).

Electrospray mass spectrometry

Electrospray mass spectrometry (ES-MS) of the proteins was performed on a Bruker Esquire 3000 Electrospray Mass Spectrometer. The protein solution was first desalted by passing a small column of Sephadex G-25, and then added with formic acid to a final concentration of 1% (v/v).

Electronic absorption spectroscopy

Electronic spectra were recorded on a Hewlett-Packard 8453 diode array spectrometer. The protein was dissolved in 100 mM sodium phosphate buffer at pH 8.0, and imidazole was added as exogenous ligand in different final concentrations if necessary. The protein concentration was 5–10 µM. Imidazole-free protein samples were obtained by a buffer-exchange column of G-25. The purified proteins in solutions exposed to air were all in their oxidized states, and the spectra of reduced proteins were recorded within 5 min after addition of a small amount of solid sodium dithionite to the ferric protein solution. Protein concentration of wild-type cyt b5 was calculated by an extinction coefficient at 413 nm ({epsilon}413 = 117 mM–1 cm–1 in its ferric state; Ozols and Strittmatter, 1964Go). The concentration of His39 mutants was determined by Bradford’s method (Bradford, 1976Go), with the wild-type cyt b5 as the control.

Imidazole titration of His39 mutants

Imidazole-free His39 mutant of cyt b5 was dissolved in 100 mM sodium phosphate buffer at pH 8.0 (~9 µM) and separated into 1.5 ml fractions. A 0.5 ml volume of concentrated imidazole solution prepared in the same buffer was added into each of the protein solutions to achieve the designed imidazole concentration. After equilibration for 10–12 h at 4°C, the electronic absorption spectroscopy of all protein solutions were determined.

DTNB assay of the His39Cys mutant

The His39Cys mutant of cyt b5 was dissolved in 1 ml of 100 mM sodium phosphate buffer at pH 8.0 with 5 mM imidazole; the protein concentration was ~60 µM. A concentrated solution of DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] was then added to a final concentration of 2.0 mM. This reaction system was equilibrated in the dark for 8–10 h at 4°C. Then the buffer was exchanged by a small column of Sephadex G-25 to the working solutions (100 mM sodium phosphate at pH 8.0 or the same buffer containing 1 mM imidazole). Finally, the electronic spectra of the reaction product, here termed His39Cys-MNB (5-mercapto-2-nitrobenzoic acid), were recorded for comparison and analysis.

Thermal denaturation

Protein samples were dissolved in 100 mM sodium phosphate buffer at pH 8.0 in the absence or presence of imidazole. The protein concentration was 5–8 µM. The changes in optical absorption at the Soret band were measured with the Hewlett-Packard 8453 diode array spectrometer equipped with a thermostated water bath. The temperature was directly measured in the cuvette holder and was maintained within ±0.1°C for 15 min to ensure that the samples reached equilibrium. Data were analyzed as previously described (Xue et al., 1999Go; Wang et al., 2000Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gene random mutagenesis, protein expression and purification

After the random mutagenesis and the screening by DNA sequencing, 16 mutant genes at His39 of cyt b5 were obtained. They are His39Ala, His39Arg, His39Asp, His39Cys, His39Gln, His39Glu, His39Gly, His39Ile, His39Leu, His39Lys, His39Phe, His39Pro, His39Ser, His39Thr, His39Try and His39Val.

Small-scale expression tests of these mutant genes were performed with IPTG induction. SDS–PAGE analysis indicated the high yields of protein peptide chains (Figure 1). Unlike the wild-type cyt b5 protein, none of the mutants could incorporate heme properly during the cell fermentation. On the other hand, it is apparent that the mutation does not affect the efficient synthesis of the apo-protein in the protease-deficient host BL21(DE3)pLysS. The target protein constitutes ~20% of the total cellular proteins.



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Fig. 1. SDS–PAGE of the expression results of some cyt b5 His39 mutants. Lane 1 and lane 10: purified wild-type cyt b5 markers; lane 2, lane 4, lane 6 and lane 8: expressions of His39Gly, His39Ser, His39Cys and His39Gln mutants with IPTG induction, respectively; lane 3, lane 5, lane 7 and lane 9: expressions of His39Gly, His39Ser, His39Cys and His39Gln mutants without IPTG induction, respectively.

 
After the protein purification and reconstitution, holo-form mutant proteins with high purity (>95%, showing a single band on SDS–PAGE) were obtained. The typical yield of the purified holo-mutants was ~30–50 mg from 10 l of culture. Imidazole was found to be very helpful for the heme reconstitution of the His39Ser mutant, imidazole-free holo-protein would lose some of its heme during the column purification processes. Imidazole also greatly changed the electronic absorption spectrum of the His39Ser mutant (see below) by competing the coordination site of heme iron in this protein, but only has a slight influence on heme reconstitution of the His39Cys mutant.

Further confirmation of gene mutagenesis and protein purification was successfully accomplished by the ES-MS (Figure 2). The measured molecular weights of cyt b5 variants are: 10634.96 ± 0.5 (wild type), 10585.87 ± 0.5 (His39Ser), and 10600.95 ± 0.5 Da (His39Cys), which correspond well to the values calculated from amino acid compositions of apo-proteins (10 634.58, 10 584.51, and 10 600.47 Da, respectively).



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Fig. 2. ES-MS result of purified His39Cys mutant of cyt b5. The measured molecular weight is 10 600.95 ± 0.5 Da.

 
Electronic absorption spectra

The electronic absorption spectra of the oxidized and reduced forms of wild-type cyt b5 and its mutants are shown in Figure 3. The absorption maxims (nm) are indicated. Electronic spectra of the wild-type cyt b5 (Figure 3A) characterize its typical bis-histidine hexa-coordination environment and low-spin heme iron in both oxidation states (Ozols and Strittmatter, 1964Go; Mathews et al., 1972aGo; Argos and Mathews, 1975Go; Sligar et al., 1987Go). The ferric His39Ser mutant (without imidazole, Figure 3B) has a spectrum that is similar to that of the wild-type met-aquomyoglobin ({lambda}max 408, 502, 630 nm; Antonini and Brunori, 1971Go) which has the high-spin ferric heme with proximal histidine and a water molecule as axial ligands. This suggests the ferric heme in this mutant is coordinated by His63 in the proximal side and by water in the distal pocket. The noticeable charge-transfer transition at 629 nm confirms the high-spin character of this oxidized mutant (Avila et al., 2000Go). The ferrous His39Ser mutant (without imidazole, Figure 3B), however, appears to be in the low-spin state, as indicated by the resolved {alpha}- and ß-bands observed in its electronic spectrum. So, Ser39 as well as His63 are considered to be coordinated to the ferrous heme iron under this condition. In the presence of imidazole (1 mM), all the oxidized and reduced His39Ser mutants show their spectra (Figure 3C) similar to that of the wild-type cyt b5, indicating that the heme is in hexa-coordinated, low-spin states in both ferric and ferrous forms. Axial ligands of oxidized His39Ser mutant protein in this condition are His63 and the exogenous imidazole. But for reduced His39Ser mutant in the presence of imidazole, while His63 is believed to be the fifth ligand, whether Ser39 or exogenous imidazole is acting as the sixth ligand can not be decided at present.



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Fig. 3. Electronic absorption spectra for oxidized (solid lines) and reduced (dotted lines) wild-type cyt b5 (A), imidazole-free His39Ser mutant (B), the His39Ser mutant with 1 mM imidazole (C), imidazole-free His39Cys mutant (D) and the His39Cys mutant with 30 mM imidazole (E). Imidazole-free His39Ser protein was measured at 10°C, other samples were examined at room temperature.

 
The electronic spectrum of the oxidized His39Cys cyt b5 (without imidazole, Figure 3D) is strikingly similar to those of the ferric CO-sensing CooA protein ({lambda}max: 362, 424, 540, 574 nm; Reynolds et al., 1998Go) and ferric cytochrome P450-imidazole complex ({lambda}max: 360, 425, 541, 578 nm; Dawson et al., 1982Go), which were reported previously to contain the low-spin heme coordinated by cysteine and histidine (or exogenous imidazole) ligands. This indicates the low-spin character and the His63-Fe(II)-Cys39 axial ligation of heme in the ferric His39Cys mutant. In addition, Cys39, the unique sulfur-containing residue of His39Cys mutant, is coordinated to the ferric heme, which is confirmed by the sulfur to Fe(III) charge-transfer bands resolved at the lower energy region (~650 and ~750 nm; Raphael and Gray, 1991Go). On the other hand, the ferrous His39Cys mutant (without imidazole, Figure 3D) shows the absorption spectrum close to that revealed by the reduced forms of human myoglobin ({lambda}max: 433, 558 nm; Adachi et al., 1991Go) and horse heart myoglobin ({lambda}max: 435, 560 nm; Lloyd et al., 1995Go) that were known to have the high-spin penta-coordinated ferrous heme. These indicate that the heme iron of the ferrous His39Cys protein is in high-spin state and only coordinated by the proximal His63 under this experimental condition. The electronic spectra of oxidized and reduced His39Cys mutant (Figure 3E) in the presence of imidazole (30 mM, details shown below) are comparable with that of the wild-type cyt b5. This leads to the conclusion that in both oxidation states, His63 and the exogenous imidazole act as two axial ligands and make the low-spin ferrous heme.

Imidazole titration and DTNB reaction of His39Cys cyt b5

To confirm the coordination between Cys39 and heme iron in ferric cyt b5 His39Cys, the protein solution was titrated with imidazole, which acts as a competitive exogenous ligand, and the result is shown in Figure 4. No significant changes of the absorption spectrum occurred when the [imidazole]/[His39Cys] ratio was <100. With the further increasing concentration of imidazole, noticeable blue shifts of the Soret band at 423 nm and the visible {alpha}/ß band at 542 nm, and a decrease of the {delta} band at about 360 nm were observed. The changes of the absorption spectra reached equilibrium at the [imidazole]/[His39Cys] ratio between 4000 and 5000, and the ferric His39Cys mutant under this condition (~30 mM imidazole and 6.7 µM protein) has the spectrum (Figure 3E) exactly similar to that of the wild-type cyt b5 (Figure 3A). In comparison, the ferric His39Ser mutant at the same concentration changes to a wild type-like spectrum (Figure 3C) before the [imidazole]/[His39Cys] ratio reaches the value of 100 (data not shown). This indicates that Cys39 in the His39Cys mutant does coordinate to the heme iron (while Ser39 in the His39Ser mutant does not have such ligation), and at high imidazole concentration, exogenous ligands can replace the sulfhydryl group and be the sixth ligand of heme. When the [imidazole]/[His39Cys] ratio exceeds 5000, a decrease of the Soret band and an increase of a broad band at about 435 nm were observed (data not shown). This shows the evidence of denaturation of this protein by excess of imidazole (Xue, 1998Go): under this condition not only the Cys39 but also the His63 were replaced by exogenous imidazole and the heme group was released from the peptide chain matrix.



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Fig. 4. Electronic absorption spectra for oxidized cyt b5 His39Cys mutant with imidazole titration measured at room temperature. The inset shows the expansion of the Soret region. The arrows indicate the absorption changes with the increment of imidazole concentration (see text). The incremental [imidazole]/[His39Cys] ratios are: 0, 50, 100, 250, 500, 750, 1000, 1500, 2000, 3000, 4000 and 5000.

 
Direct evidence of the coordination between Cys39 and heme iron in the ferric His39Cys mutant is also obtained from the DTNB assay (Figure 5). After the reaction with DTNB, the specific oxidant of the sulfhydryl group (Ellman, 1959Go; Vasák, 1991Go), the resultant His39Cys-MNB complex shows the electronic spectra similar to that of the His39Ser mutant (Figure 3B and C) at the Soret band and visible regions in the absence or presence of imidazole (1 mM only), respectively. These transformations are unquestionably caused by the cut-off of the coordination bond between Cys39 and heme iron.



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Fig. 5. Electronic absorption spectra for oxidized (solid lines) and reduced (dotted lines) cyt b5 imidazole-free His39Cys-MNB complex (A, measured at 10°C), and His39Cys-MNB with 1 mM imidazole (B, examined at room temperature). His39Cys-MNB complex is the product from the His39Cys mutant of cyt b5 after reaction with DTNB.

 
Thermal denaturation studies

To quantify the decrease of heme-binding stability caused by the His39 replacement in the His39Ser and His39Cys mutants of cyt b5, thermal denaturation studies were performed and monitored by the UV-visible absorption changes of the protein’s Soret band, reflecting the strength of heme binding directly. The normalized denaturation curves of the wild type and mutants of cyt b5 in their oxidized forms as the function of temperature are shown in Figure 6A. An example of absorption spectra of the thermal denaturation of the ferric His39Cys mutant are shown in Figure 6B. The thermal denaturation curves indicate the two-state processes as described by Pace (Pace, 1986Go). The isosbestic points observed in denaturation spectra show no detectable intermediates in the unfolding process. Transition temperature (Tm), enthalpy change ({Delta}Hm), entropy change ({Delta}Sm) and difference in free energy change ({Delta}{Delta}GD50%) are summarized in Table I. Based on these values, it is obvious that the heme-binding stabilities of all heme axial mutants of cyt b5 are significantly lower than that of the wild-type protein even in the presence of the exogenous ligand of imidazole.



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Fig. 6. (A) Thermal denaturation curves of oxidized cyt b5 variants: (closed square) wild type, (closed circle) imidazole-free His39Ser mutant, (open square) the His39Ser mutant with 1 mM imidazole, (open circle) imidazole-free His39Cys mutant, and (closed triangle) His39Cys-MNB complex with 1 mM imidazole. The data for Soret absorbance were normalized. (B) Electronic absorption spectra for the thermal denaturation of oxidized imidazole-free His39Cys mutant of cyt b5. The arrows indicate the absorption changes as the temperature is increased.

 

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Table I. Thermodynamic data for thermal denaturation of cyt b5 and its mutantsa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutation strategy and mutant protein selection

Cyt b5 polypeptide chain holds heme using two axial histidine ligands, His39 and His63, which are located in the centers of two face-to-face heme-binding loops. In the protein crystal structure of bovine microsomal cyt b5 (Durley and Mathews, 1996Go), the loop including His39, exhibits a more flexible structure compared to the other loop including His63. The coordination bond distance between the nitrogen atom of His39 and the heme iron (~2.07 Å) is slightly longer than that between His63 and the heme iron (~2.00 Å). The previous NMR study on the rat microsomal apo-cyt b5 also revealed that the His63 loop shows a larger fluctuation than the His39 loop after the heme dissociation (Falzone et al., 1996Go). Thus, the interaction of the His39 loop with heme seems to be relatively weaker than that of the His63 loop in the microsomal cyt b5, and alteration of the His39 ligand would incur relatively less damage to the protein stability. So we chose His39 as the mutation site in this work. The above design consideration was also confirmed by the recently published paper on the His39Leu and His63Leu variants of the rat microsomal cyt b5 (Ihara et al., 2000Go), in which the overall equilibrium heme association constant of the His39Leu mutant was found to be 29 times larger than that of the His63Leu mutant. The authors also mentioned that without exogenous ligands, the His39Leu mutant forms an aquomet-species at 10°C and the His63Leu variant is absolutely unstable.

As mentioned in the Introduction, mutation on the heme axial ligand of cyt b5 would lead to substantially decreased heme incorporation stability because of the relatively low interaction of the protein peptide chain with heme and the essential dependence of heme-holding stability on the bis-histidine axial ligation. To screen out some heme axial mutant proteins with higher heme-binding stability, and to construct an axial mutants library making a good base for further work, we have performed a systematic random mutagenesis at His39 in bovine liver microsomal cyt b5. However, none of the resultant mutated genes incorporate heme during cell fermentation. This indicates that the decrease of heme binding stability caused by the alteration of heme axial ligands in cyt b5 is a common phenomenon indeed. The almost equal yield of mutated apo-protein during expression implies that these apo-proteins might have similar stabilities and structures. This structural stability should be resulted from the existence of the structure-keeping domain in the protein, i.e. core 2, which was observed to be stable and intact in the wild-type apo-cyt b5 (Falzone et al., 1996Go).

From the mutated gene library of cyt b5, we chose the His39Ser and His39Cys mutants, which have the hydrophilic amino acid residue substitution at His39, to be expressed into proteins first. These two residues possess the potential of being coordinated to the heme iron using the oxygen (Ser39) or sulfur (Cys39) atoms, and such coordination would deserve stabilization of the resultant holo-proteins. This was indeed found to be the case in this work. At the same time, Ser and Cys are quite similar in their molecular structures and volumes; this makes the comparisons of their experimental results more meaningful.

More significantly, an important goal of our studies is, however, to examine the possibility of creating a new protein molecule with catalytic reactivity by artificial modification of an electron transfer hemoprotein (such as cytochrome b5). This kind of study would also aid in finding the key structural differences between various hemoproteins having distinct natural functions. Reasons for the selection of these hydrophilic amino acid residues to replace His39 in cyt b5 also include an important consideration of engineering ‘a hydrophilic heme catalytic pocket into cyt b5’, which is essential for binding of substrate and creating the catalytic activity of the protein. And this has been found indeed to be the case (Wang et al., 2002Go).

Variation of heme coordination environments in the His39Ser and His39Cys mutants of cyt b5

It is noteworthy that most of the published cyt b5 heme axial mutants (without any exogenous ligands) show similar experimental results with the cyt b5 His39Ser mutant in this work. The microsomal cyt b5 His39Met (Sligar et al., 1987Go) and outer mitochondrial membrane (OM) cyt b5 His63Met (Rodríguez and Rivera, 1998Go) mutants all have absorption spectra very similar to that of our His39Ser mutant in both oxidation states. In addition, these mutants all were reported to be in a hexa-coordinated high-spin state in the ferric form and hexa-coordinated low-spin state in the ferrous form, respectively. Although it was not concluded whether Met39 or water is the sixth ligand of ferric microsomal cyt b5 His39Met mutant (Sligar et al., 1987Go), the coordination of Met63 to heme was clearly ruled out by NMR results, and water was believed to be the distal ligand in ferric OM cyt b5 His63Met mutant (Rodríguez and Rivera, 1998Go). These results are consistent with the conclusion of the His39Ser mutant presented here.

A unique characteristic of the His39Cys mutant is the ability of the substitutional Cys39 to coordinate to the heme iron in the oxidized state, no other heme axial mutant of cyt b5 shows such coordination up to now. Not only the sulfur to Fe(III) charge-transfer bands at ~650 nm and ~750 nm, but also the well-resolved symmetrical {delta} band at ~360 nm provides clear evidence of the coordination between Cys39 and heme in ferric cyt b5 His39Cys mutant. In some hemoproteins such as cyt b5, cytochrome c, cytochrome P450 and myoglobin, the {delta} band is always weak in intensity and asymmetric, or even just a small shoulder. Such a symmetrical {delta} band with a relatively high peak intensity is the common observation in several variants of hemoproteins having the histidine (or imidazole)-cysteine (or sulfhydryl) coordination environment, such as the natural CO-sensing CooA protein (Reynolds et al., 1998Go), cytochrome P450-imidazole complex (Dawson et al., 1982Go; Sono et al., 1986Go) and the Met80Cys mutant of cytochrome c (Raphael and Gray, 1991Go). This is a similar situation to the ligation existing in cyt b5 His39Cys mutant. It suggests that such a profile of the {delta} band should be assigned to the His-Fe(III)-Cys coordination.

In the presence of imidazole, resultant absorption spectra of the cyt b5 His39Ser mutant both in oxidized and reduced states are very similar to those obtained from wild-type cyt b5. This indicates that in the ferric form of this mutant the exogenous imidazole readily replaces the distal water molecule, and couples with the intact His63 to mimic the bis-histidine hexa-coordination environment of the wild-type protein. At present it is not known whether Ser39 or imidazole is the sixth ligand in the ferrous His39Ser mutant under this condition because of the close similarity between its spectra in the presence and absence of imidazole.

In comparison with the facile ligation of the exogenous imidazole to heme observed in the ferric His39Ser mutant, the ferric cyt b5 His39Cys mutant showed strong resistance against such imidazole replacement. This testifies to the existence of a strong endogenous sixth ligand of heme. At quite a high imidazole concentration, the ferric His39Cys mutant just showed the spectrum (Figure 3E) partially similar to that of wild-type cyt b5. The only slight red-shift of the Soret band (414 nm for ferric His39Cys mutant under this condition and 413 nm for wild-type cyt b5) and the relatively broader Soret band of the His39Cys mutant under this condition in comparison with those of the wild-type cyt b5 imply that there is still a competitive equilibrium for sixth coordination of the heme iron between the endogenous ligand and exogenous imidazole. The disappearance of sulfur to Fe(III) charge-transfer bands and the obvious decrease of {delta} band during imidazole titration all declare the coordination between Cys39 and heme iron in this ferric mutant. (Full agreement is also directly obtained from the reactions of the His39Cys mutant with DTNB.)

Heme-binding stability of cyt b5 His39Ser and His39Cys mutants

Thermal denaturation is a useful method for studying protein stability. Monitored by the changes in optical absorption at the Soret band, the heme dissociation course can be directly displayed during the thermal denaturation process. From the parameters summarized in Table I, it is evident that the heme-binding stabilities of His39 variants of cyt b5 are significantly lower than the wild-type protein.

The heme-binding stability of cyt b5 and its mutants in their oxidized states has the order: wild type >> His39Cys > His39Ser, that corresponds to the different heme coordination environments of these proteins. The His39Ser mutant has a Tm value about 40°C lower than that of the wild-type cyt b5. The His39Cys mutant has a conceivably higher Tm value than the His39Ser variant, but still >20°C lower than the wild-type protein. This indicates that the endogenous sixth ligand of heme favors the heme incorporation, and the inherent histidine seems to be the best. A quite increased stability of the His39Ser mutant in the presence of imidazole shows that imidazole can distinctly strengthen the interaction between the polypeptide chain and heme in this protein, even if it is an exogenous ligand of the heme iron.

Interestingly, it was found that the order of the heme-binding stability of the wild-type cyt b5 and its mutants is the reverse of their peroxidase-like activities (Wang et al., 2002Go), which is similar to the result reported for the synthetic amphiphilic short peptide-heme complexes (Sakamoto et al., 1999Go). All this suggests that the open site in the heme pocket is necessary for gain of the catalytic reactivity of hemoproteins.

It is remarkable that there is just one atom (oxygen or sulfur) difference between the cyt b5 His39Ser and His39Cys mutants, although there is somewhat a difference in hydrophobicity between Ser and Cys residues (Richardson and Richardson, 1989Go), these two mutants exhibit greatly distinct properties as shown above. No doubt, difference in the coordination ability between two residues is one of the main factors governing the distinction. It is the most desire to have the three-dimensional structures for these variants. We are now trying to get the protein structures by X-ray crystallography. The cyt b5 His39Cys mutant is the first object because of its outstanding characters and relatively higher stability.


    Acknowledgements
 
It is a pleasure to acknowledge Professor A.G.Mauk of the University of British Columbia, Canada, for his kind gift of cytochrome b5 gene. This work was supported by National Natural Science Foundation of China.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received April 11, 2003; revised October 13, 2003; accepted October 23, 2003





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