Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Abstract

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

Chloroperoxidase (CPO; EC 1.11.1.10) is the most versatile catalyst in the hemoprotein family. CPO possesses a broad spectrum of oxidative activities, including those characteristics of heme peroxidases, catalases, and the P450 cytochromes. CPO shares with horseradish peroxidase and other members of the plant peroxidase family the ability to catalyze one- and two-electron peroxidations (1). It was originally discovered as a peroxidative chlorination catalyst involved in the biosynthesis of caldariomycin (2, 3). CPO behaves as a catalase in terms of catalyzing the dismutation of hydrogen peroxide and the oxidation of alcohol (1). It mimics P450 cytochromes in catalyzing heteroatom dealkylation (4, 5), benzylic hydroxylations (6), and oxygen transfer to alkenes (7–9), alkynes (10, 11), sulfides (12, 13), and arylamines (14, 15). CPO is especially adept in the stereoselective epoxidation of alkenes (7–9, 16, 17) hydroxylation of alkynes (10, 11) and in the production of chiral sulfoxides (12, 13).

CPO contains the heme iron prosthetic group found in most other peroxidases, but in contrast to other peroxidases, CPO does not have a proximal histidine heme iron ligand and instead shares with the P450 cytochromes the property of having a proximal heme iron thiolate ligand (18–25). CPO also is unique in the hemoprotein family by virtue of having a glutamic acid residue distal to the heme-iron (25). It has been postulated that this glutamic acid residue functions as an inflexible acid-base catalyst and plays a mechanistic role similar to that played by the distal histidine in traditional peroxidase chemistry (26). Obviously, the unique active site structural characteristics of CPO must account for the versatility of CPO as an oxidation catalyst. These considerations prompted this investigation into CPO structure-function relationships.

In this paper, we report the replacement of Cys-29, the heme iron proximal ligand in CPO, with a histidine residue. This was accomplished by site-directed mutagenesis followed by expression in the fungus, Caldariomyces fumago. C. fumago also produces wild-type CPO and was considered as an expression vector for CPO mutants only after extended attempts with established expression systems met with very limited success. Wild- type CPO contains a relatively high degree of posttranslational modifications. There are 15 glycosylation sites in wild-type CPO (25), plus one important disulfide bond (27). The lack of both glycosylation and disulfide bond formation may contribute to the problem of expression in Escherichia coli. Because C. fumago also produces wild-type CPO, the transformation event required that the mutant gene replace its normal chromosomal copy. The gene replacement event was maximized by using constructs that promoted double crossover events.

Materials and Methods

Restriction endonucleases and most other enzymes used in vector construction and molecular biology were obtained from GIBCO/BRL and New England Biolabs. Pfu DNA polymerase and E. coli NM 522 competent cells were from Stratagene. DNA plasmid purification kits were obtained from Qiagen (Chatsworth, CA) and were used according to the supplier’s instructions.

Vector Construction.

The pTHC vector initially used in the C. fumago transformations contained a hygromycin B phosphotransferase (hph) resistance gene and the mutant CPO gene. It was constructed by inserting a 3-kb EcoRI/XbaI fragment from plasmid pDH 25 into pTZ19R (28) to produce a high-copy number shuttle vector (29). The EcoRI/XbaI fragment contains the hph gene under the control of the trpC promoter and terminator (Genentech). The CPO gene together with its promoter sequences was introduced into pTHC by placing an EcoRI restriction site upstream from the CPO promoter and a BamHI site downstream from the CPO coding sequence. Pairs of PCR primers were used to introduce the ClaI site upstream from the start codon of the CPO gene. The pTHC plasmid constructed in this manner contained a copy of the 900-bp CPO promoter DNA sequence, the CPO coding region, and a trpC terminator. To improve integration via homologous recombination, a modified vector, pTHN, was constructed from pTHC (Fig. 1). In pTHN, a copy of approximately 200 bp of the 3′ untranslated region of the CPO gene was inserted into the XbaI site upstream from the SphI site that was used for linearizing the plasmid (30). All PCR reactions were performed following the Kolmodin and Williams procedure, including usage of “Hot-start” and “Touch-down” techniques to eliminate nonspecific PCR product (31). The PCR products were purified on a Qiagen column.

Figure 1.

E. coli/C. fumago shuttle vector pTHN. The shuttle vector was derived from pDH25 and pTZ19R. It contains a selectable β-lactamase (Amp′) gene and a selectable hygromycin B (hyh) gene. The plasmid encodes a mature CPO coding region, a 200-bp sequence from the 3′ untranslated region of the CPO gene and native promoter sequences.

Site-Directed Mutagenesis.

Oligonucleotide site-directed mutagenesis was performed following the Quickchange procedure of Stratagene by using Pfu polymerase. The mutagenic oligonucleotide primers encoded a Cys-29 → His mutation and a silent mutation, which removed a BssSI site from the CPO gene. The BssSI site was removed to provide a convenient method to screen for the presence of the mutant genome. The primers were purified by PAGE. A cDNA sequence encoding a full-length wild-type CPO, previously cloned in this laboratory (32) in vector pTZ19R, served as the template for PCR amplification. This PCR product was transformed into E. coli NM 522 competent cells. The transformants were selected on Luria-Bertani (LB) agar plates containing 50 mg/liter ampicillin. Recombinant colonies were screened by restriction mapping and verified by DNA sequencing. The mutant CPO DNAs were digested with BamHI/ClaI and subcloned into shuttle vector pTHN.

Preparation of Spheroplasts.

C. fumago spheroplasts were prepared by using a modification of the procedure developed by Patterson (33). Fresh mycelia were grown in 100 ml of fructose culture media (34) at 21°C for 5 days on a shaker. The mycelia were harvested by centrifugation and the pellet was resuspended in 50 ml of 0.5 M KCl and 0.1 M Mes, pH 5.0. After washing twice, the mycelia were suspended in 50 ml of the same buffer solution and Novozyme 234 was added to a final concentration of 5 mg/ml. The suspension was gently rocked at room temperature for 2 h. Cell debris was removed by centrifugation at 80 × g for 5 min. The supernatant fraction was filtered through a sterile 10-ml plastic pipette tip containing loosely packed glass wool to remove any remaining cellular debris. The spheroplasts were collected by centrifugation at 1,000 × g for 5 min. The pellet was washed twice with 0.8 M sorbitol and 10 mM bis-Tris buffer, pH 6.5. Each 100-ml culture yielded 5–10 × 10⁷ spheroplasts.

Transformation.

The transformation procedure also was based on the procedure developed by Patterson (33). Ten microliters of linearized pTHN (5 μg/μl) was mixed with 1 × 10⁷ spheroplasts (100 μl) in 0.8 M sorbitol, 50 mM CaCl₂, and 10 mM bis-Tris buffer, pH 6.5. The mixture was incubated on ice for 30 min. Then 1.5 ml of a 30% solution of polyethylene glycol (molecular weight 3,325), 50 mM CaCl₂, and 10 mM bis-Tris, pH 6.5 was added with gentle stirring. The spheroplasts were incubated at room temperature for 40 min. This step was repeated for a second 40-min incubation. After the second incubation, 10 ml of a 1 M solution of sorbitol was added and the mixture was centrifuged at 1,000 × g for 5 min. The transformed spheroplasts were recovered in the pellet fraction. The spheroplasts were resuspended in 10 ml of the hygromycin selection medium (1 M sorbitol, 4% glucose, 0.5% malt extract, and 0.2% each of NaNO₃, KH₂PO₄, and MgSO₄ supplemented with 0.75 mg/ml hygromycin B and 0.005% deoxycholate) and were transferred to a plastic Petri dish. Antibiotic-resistant colonies adhering to the bottom of the Petri dish appeared on the fourth day of incubation at room temperature. To obtain stable transformants, a second round selection was required. Positive clones from the second round selection were picked and transferred to 24-well plates containing fructose culture medium (34). The plates were incubated with shaking at room temperature for 7 days.

Detection of Mutants.

After 7 days of growth in the 24-well plates, aliquots of the medium from each well were collected and assayed for chlorination activity and peroxidation activity. Aliquots of mycelia also were collected from each well for DNA extraction and were washed with 0.1 M phosphate buffer, pH 5.0. The washed mycelia were mixed with 200 μl of lysis buffer (50 mM Tris⋅HCl, pH 7.5, 1% SDS, and 1 mM α-mercaptoethanol) and one volume of glass beads and blended at 6,000 rpm to break open the mycelial cells. After breakage, the crude extract was boiled for 10 min and DNA was extracted by using the phenol-chloroform procedure and collected by isopropanol precipitation. The DNA isolated from each of the clones was used as a template for a 35-cycle PCR amplification. In the PCR screening, pfu polymerase was used instead of Taq polymerase. After purification of the PCR products by using a Qiagen column and digestion with BssSI, aliquots of each of the PCR products were subjected to electrophoretic separation on 3% NuSieve agarose gels. Wild-type DNA gave two bands in the agarose gels, whereas the mutant DNA gave a single band. Those clones whose DNA showed single bands were scored mutation positive and were collected for further analysis. The amplified nucleotide sequence encoding the mutant CPO was confirmed by automated dideoxy sequencing and it was then used for recombinant mutant CPO expression. The mutant enzymes were further analyzed by examining the visible absorption spectra of their ferrous–CO complexes. Spectra were recorded on a Shimadzu UV-1201 spectrometer. Extinction coefficients for the mutant CPO were determined by the pyridine-hemochrome (35) and formic acid methods (36).

Expression and Purification of Mutant CPO.

The purification of the mutant enzyme was carried out by the Morris and Hager protocol (3) with minor modifications. Acetone rather than ethanol was used in the solvent fractionation step. After acetone precipitation, the mutant protein was purified on a DEAE Sephadex (Pharmacia Fine Chemicals) column equilibrated with 0.05 M potassium phosphate buffer, pH 6.0. The mutant CPO was eluted from the column by using a linear ionic strength gradient prepared by placing 300 ml of 0.05 M potassium phosphate buffer, pH 6.0 in the mixing chamber, and 300 ml of 0.2 M potassium phosphate buffer, pH 6.0 in the reservoir. The mutant CPO was collected, dialyzed against 0.05 M phosphate buffer, pH 5.0, and then concentrated by using a Centricon-30 ultrafiltration concentrator (Amicon). The Reinheitzahl value (Rz) was used to check the purity of the final sample. The Reinheitzahl value number for highly purified preparations of both wild-type (398/280 nm) and mutant (402/280 nm) CPO were in the 1.4–1.6 range.

Enzyme Activity.

Chlorination activity was quantified in the monochlorodimedone (MCD) assay (37). Peroxidation activity was measured by using 2,2′-azino-bis-3-ethy-benzthiazoline-6-sulfonic acid (ABTS) as the electron donor (38). The catalase activity for wild-type and mutant CPO was based on measuring the rate of formation of molecular oxygen by using an oxygen electrode (1).

Epoxidation Activity.

Initial rates of styrene epoxidation activity were determined by following the decrease in absorption at 262 nm as a function of time in a reaction mixture containing approximately 5 μg of native or mutant CPO, 2 mM H₂O₂, and 300 μM styrene in 10 mM citrate buffer, pH 5.5 in a total volume of 3 ml. Two olefinic substrates, styrene and cis-β-methyl styrene were used to compare product yield and the enantioselectivity shown by the native and mutant CPO. For these assays, a reaction mixture was prepared which contained 70 μl of styrene or cis-β-methyl styrene, 100 μl of tert-butyl hydrogen peroxide, and 400 μl of dimethyl formamide or tert-butanol. In the assay, 25 μl of the above mixture was added to 2 mg of wild-type or mutant CPO in 750 μl of 100 mM citrate buffer, pH 5.5. The reaction mixture was incubated at room temperature for 45 min. After incubation, the reaction was stopped by the addition of 50 μl of 1 M Na₂S₂O₃ in a saturated solution of NaHCO₃. The reaction mixture was extracted with 300 μl of isooctane containing undecane as an internal standard. The extracted organic phase was analyzed by gas chromatography (capillary column DB-5, 25°C injector and FID at 250°C, column 100°C, helium at 1 ml/min) to measure substrate disappearance and product formation. HPLC analysis of the organic phase on a Pirkle Concept WhelkO-1-SS column (2% isopropyl alcohol in hexane, 1 ml/min, UV detector at 254 nm) was used to determine the enantiomeric excess of epoxides formed in the reaction.

Results and Discussion

C. fumago Expression System.

A diagram of the E. coli/C. fumago shuttle vector which was used to express the C29H mutant is shown in Fig. 1. This vector gave a relatively low yield of positive clones (≈10%); however, those clones which contained the replaced wild-type gene produced active mutant CPO in high yield (equivalent to the yield of native CPO). The remaining 90% of the clones obtained in the transformation experiments containing both wild-type and mutant genes were not particularly useful. Previous attempts in this laboratory to express recombinant CPO in E. coli (39) or other expression systems gave relatively unsatisfactory results (29, 33, 40). Large amounts of apoCPO can be produced in E. coli in the form of denatured inclusion bodies; however, reconstitution of holoCPO from the inclusion body CPO with iron protoporphyrin IX met with very limited success (39). Approximately 5% reconstitution was the maximum level of holoCPO recovery in experiments that used both dialysis and high pressure techniques to aid in the renaturation process. Because wild-type CPO is produced by C. fumago at levels of grams per liter as a secreted enzyme, it was tempting to explore the fungus itself as an expression vector. Pioneering work by Patterson (33), Zong (29), Sigle (40), and Allain (41) indicated that C. fumago showed promise as an expression vector; however, this early work also showed that deletion knockouts of wild-type CPO were lethal events. Subsequent experiments have shown that transformation of C. fumago and expression of the directed evolution CPO mutants can be readily performed under conditions that lead to the incorporation of the mutant DNA sequences via single crossover events. Under these conditions, the mutant gene is inserted into the genome in parallel with the wild-type gene. To distinguish and separate the two populations of expressed CPOs, a flag epitope (41) or a six-histidine peptide tag (40) was employed. Although successful in the sense that mutant CPOs could be detected and isolated by using these inserts, it also was apparent that the use of the tagged peptide sequences greatly diminished the yield of recombinant CPO. We thus turned to gene replacement as a more suitable technique for the isolation of site-directed mutants of CPO. Development of techniques for gene replacement is important because they serve as a mechanism for the evaluation of the effect of mutations in the absence of spurious position influences. The availability of C. fumago as a high-level expression system for CPO now allows a direct examination of the role of active-site amino acid residues in this enzyme. In these experiments, the original vector, developed by Zong (29), was modified by adding DNA sequences that increased the degree of homology between the mutant and wild-type flanking DNA sequences. Waldman and Liskay (42) showed that an uninterrupted terminal homology of 130 bp was a minimal requirement for gene replacement. Our observations are consistent with their findings. The addition of a 200-bp sequence from the 3′ untranslated region of the CPO gene significantly increased the opportunity for recombination. Besides the demand for extended homologous stretches, the probability for gene replacement also is species dependent. For instance, gene replacement has been successful in Aspergillus nidulans (43), but difficulties have been encountered when applying these same techniques to Neurospora crassa (44, 45). Our experiments have shown that gene replacement in C. fumago is quite feasible.

Visible Absorption Spectra.

Heme proteins have characteristic visible Soret absorption bands that convey significant information about the structure and coordination state of their active sites. Fig. 2 compares the spectra of the ferrous–CO complexes of native CPO and the Cys-29 → His mutant. The Soret peak for the ferrous–CO complex of wild-type CPO is at 446 nm, whereas the mutant Soret peak is found at 420 nm. These visible absorption data are consistent with the conclusion that an imidazole nitrogen of histidine has replaced the thiolate ligand found in wild-type CPO. Stern and Peisach (46) first showed that a long wavelength Soret absorption band signifies the presence of a thiolate heme iron ligand in heme proteins. The presence of thiolate ligands in native CPO and P450 cytochromes has been confirmed by extensive spectroscopic techniques (22, 47–50) and x-ray crystallographic analysis (25, 26). The Soret band for the ferrous–CO complexes of heme proteins containing an imidazole proximal ligand centers at 418–420 nm (51). The Soret peak of the ferrous–CO complex of the Cys-29 → His mutant is at 420 nm, and is consistent with the presence of a histidine-imidazole ligand. The ferrous–CO complex of the alkaline denatured form of CPO (CPO420) also has this 420 nm absorbance band; however, in this case, the 420 species is inactive (52).

Figure 2.

Visible spectra of the ferrous–CO complexes of wild-type CPO–CO (– – –) and the C29H mutant (———) in 0.05 M potassium phosphate buffer, pH 5.0.

The optical absorption spectra of the mutant and native CPO and the spectra of their respective azide complexes are recorded in Figs. 3 and 4. Fig. 3 compares the spectrum of native CPO with the Cys-29 → His mutant. The Soret band for native CPO is at 398 nm and the corresponding band for the mutant is at 402 nm. Fig. 4 compares the spectra of the azide complexes of native and mutant CPOs. The fact that there is a small shift in the Soret peak in the azide–mutant complex suggests that the binding of azide to the heme prosthetic group in the mutant has been altered. This change could be caused by a slightly altered conformation of the complex with increased electron repulsion in the dz² orbital. This change also may reflect a weakened iron-imidazole ligand. It has been reported that cyanide (binding characteristics similar to azide) binds more tightly to P420cam than P450cam (53). P420cam has lost the thiolate ligand present in the P450 form. Our data suggest that His-29 in the mutant CPO is able to correctly coordinate with the heme iron and allow the mutant to function in an equivalent fashion in comparison with the wild-type enzyme.

Figure 3.

Absorption spectra of the C29H mutant (———) and native CPO (– – – ). The enzymes were diluted in 0.05 M potassium phosphate buffer, pH 5.0.

Figure 4.

The optical spectrum of mutant (———) and native CPO (– – –) azide complexes. The spectra of the azide derivatives were measured in 0.05 M phosphate buffer, pH 5.0, and 0.56 M sodium azide.

Enzymatic Characterization of the Cys-29 → His Mutant Enzyme.

A comparison of the chlorination, epoxidation, peroxidation, and catalase activity of the native and mutant CPOs is shown in Table 1. The data show that the Cys-29 → His mutant has approximately 60–75% of the native CPO activity in all four reactions. Previous hypotheses concerning the role of Cys-29 in CPO chemistry were based on the assumption that the strong electron-donating contribution of the Cys-29 thiolate ligand was an important component of CPO catalysis and ligand binding (54–56). In the case of cytochrome P450, Dawson et al. (48, 55, 57) have suggested that the role of the thiolate ligand, as a strong electron donor, is to destabilize and facilitate heterolytic O—O bond cleavage and thus promote the formation of the oxoiron (IV) porphyrin π-cation radical (compound I). It has also been postulated that the presence of the thiolate ligand in CPO accounts for the ability of this enzyme to function as a chlorination catalyst by virtue of conferring a low oxidation-reduction potential on the enzyme. Other peroxidases, having a histidine rather than a thiolate ligand, are able to oxidize bromide and iodide but not chloride. Because the Cys-29 → His mutant has only lost about 25% of its chlorination activity, the thiolate ligand hypothesis is no longer tenable. Instead, the results with the Cys-29 → His mutant suggest that the amino acid residues on the distal side of the heme play the important role or roles in promoting the versatility of this enzyme as an oxidation catalyst. These considerations focus attention on the unique distal Glu-183 residue in CPO. It has been postulated that Glu-183 provides additional electrostatic destabilization to the oxoferryl π-cation intermediate, thus promoting the formation of the highly reactive Compound I intermediate capable of oxidizing chloride at a low pH (26). Sundaramoorthy et al. (26) propose that Glu-183 serves as general acid-base catalyst and first reacts with the hydrogen peroxide-heme adduct as a general base to abstract a proton from the adduct and generate a hydroperoxoiron complex. In the second step, they suggest that the protonated form of Glu-183 serves as a general acid to facilitate O—O bond cleavage.

Table 1.

Comparison of the kinetics of native and C29H mutant CPO

Reaction	Substrate concentration	Pseudo 1st order rates mol of product/s/mol of enzyme		Ratio
		Wild type	Cys-29 → His	Cys-29 → His/wild type
Chlorination	0.17 mM MCD (a)	850	691	0.81
Peroxidation	10 mM ABTS (b)	0.169	0.144	0.85
Epoxidation	0.3 mM styrene (c)	4.8	3.7	0.77
Catalase	0.5 mM H2O2 (d)	1500	900	0.60

All assays were done at room temperature. Initial rates were determined from the linear portion of the time curve. In addition to substrate, the assay reaction mixtures contained (a) 20 mM KCl, 10 nM CPO, 2 mM H₂O₂, and potassium phosphate buffer, pH 2.75; (b) 2.3 μM CPO, 2 mM H₂O₂, and 100 mM potassium phosphate buffer, pH 6; (c) 40 nM CPO, 2 mM H₂O₂, and 100 mM potassium citrate buffer, pH 5.5; (d) 464 nM CPO and 50 mM potassium phosphate buffer, pH 5. 

Halogenation Activity of the Cys-29 → His Mutant.

The Cys-29 → His mutant is active as a halogenation catalyst. The mutant shows about 75% of the wild-type activity in the monochlorodimedone assay. The pH optimum for chlorination has shifted to a slightly higher pH value. The optimum pH for chlorination by native CPO is 2.75, whereas the optimum for the mutant CPO is 3.25 (data not shown).

Epoxidation Activity of the Cys-29 → His Mutant.

Table 1 compares the epoxidation activity of the mutant and wild-type CPO. The epoxidation activity of the Cys-29 → His mutant is approximately equivalent to the epoxidation activity of wild- type CPO in terms of kinetic, product yield, and enantioselectivity. The epoxidation of styrene and cis-β-methyl styrene by the mutant gives essentially wild-type oxidation rates. The yields of product and enantiomeric excess are virtually the same as found with native CPO.

These experiments have tested for the first time the importance of a thiolate-heme ligand in oxidative enzyme catalysis. Previous attempts to replace the cysteine-thiolate ligands in P450cam and cytochrome P450_d were not successful apparently because of the instability of the apoP450s. In both cases, the investigators were unable to reconstitute the apoP450 with heme to yield the holoenzyme (58, 59). The experimental results presented in this paper demonstrate that, in contrast to accepted wisdom, the thiolate ligand is not required for P450-type reactions. The only related results on the role of proximal ligands comes from the cavity complementation mutants in cytochrome c peroxidase. Choudhury et al. (60) have examined the role of the proximal ligand in cytochrome c peroxidase catalysis where the native histidine ligand has been replaced with glutamine, glutamate, and cysteine. The glutamine and glutamate mutants retain high levels of activity; however, the His-175 → Cys mutant loses approximately 93% of the native peroxidase activity. Choudhury et al. (60) attribute the loss of activity to the oxidation of the mutated cysteine to a cysteic acid residue; thus, in terms of catalysis, the proximal ligand in both CPO and cytochrome c peroxidase is not an important active-site residue.

Adachi et al. (61) have replaced the proximal histidine ligand of human myoglobin with cysteine and examined the spectroscopic and enzymatic activities of this modified globin. They find that Cys-myoglobin has an enhanced rate of heterolytic O—O cleavage of cumene hydroperoxide, a fivefold increase in epoxidation activity in comparison with wild-type myoglobin and a lower redox potential. These results indicate that the mutant myoglobin becomes more like CPO; however, it is difficult to compare the myoglobin results with CPO catalysis because of the vast difference in rates of reaction. The pseudo first-order rate constant for the epoxidation of styrene by CPO is 48 mol s⁻¹ mol⁻¹ and based on the data provided by Adachi et al.(61), we estimate the first-order rate constant for myoglobin to be 1.5 × 10⁻⁴ mol s⁻¹ mol⁻¹; thus, even with a fivefold increase in the rate of epoxidation by the cis-myoglobin, the epoxidation of styrene by myoglobin is approximately five magnitudes lower than the CPO epoxidation rate. The concentration of myoglobin and mutant myoglobin used by Adachi et al. (61) in their styrene epoxidation reactions is 10- to 50-fold higher than the amount of epoxide product formed in the reaction. Under these circumstances, it would appear that myoglobin is more a reactant than a catalyst and brings into question any interpretation of the effect of the mutation on catalysis.

In a recent report, Ortiz de Montellano et al. (62), has shown that a heme oxygenase loses its native oxygenase activity and is converted to a NADH oxidase when the proximal histidine heme ligand is converted to a cysteine.

Future experiments will have to increase knowledge of many aspects of CPO catalysis by the Cys-29 → His mutant. A priori, it would appear that compound I formation should take place at normal or nearly normal rates, but this will have to be verified by experiment. The role of Glu-183 in CPO catalysis must be examined because this active site amino acid becomes the obvious candidate for offering an explanation for the unique activity of this enzyme. Likewise, the role of His-105 and Asp-74 in providing a proton shuttle to Glu-183 needs to be examined. The development of C. fumago as an expression vector will allow us to provide answers to these questions.

Abbreviation

CPO

chloroperoxidase