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Peroxygenase activity of cytochrome c peroxidase and three apolar distal heme pocket mutants: hydroxylation of 1-methoxynaphthalene.

Erman JE, Kilheeney H, Bidwai AK, Ayala CE, Vitello LB - BMC Biochem. (2013)

Bottom Line: Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044 ± 0.0001 min-1.Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity.Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.

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ABSTRACT

Background: The cytochrome P450s are monooxygenases that insert oxygen functionalities into a wide variety of organic substrates with high selectivity. There is interest in developing efficient catalysts based on the "peroxide shunt" pathway in the cytochrome P450s, which uses H2O2 in place of O2/NADPH as the oxygenation agent. We report on our initial studies using cytochrome c peroxidase (CcP) as a platform to develop specific "peroxygenation" catalysts.

Results: The peroxygenase activity of CcP was investigated using 1-methoxynaphthalene as substrate. 1-Methoxynaphthalene hydroxylation was monitored using Russig's blue formation at standard reaction conditions of 0.50 mM 1-methoxynaphthalene, 1.00 mM H2O2, pH 7.0, 25°C. Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044 ± 0.0001 min-1. Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity. The highest activity was observed for CcP(triAla), a triple mutant with Arg48, Trp51, and His52 simultaneously mutated to alanine residues. The turnover number of CcP(triAla) is 0.150 ± 0.008 min-1, 34-fold greater than wild-type CcP and comparable to the naphthalene hydroxylation activity of rat liver microsomal cytochrome P450. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, CcP(triAla) is inactivated within four cycles of the peroxygenase reaction.

Conclusions: Protein engineering of CcP can increase the rate of peroxygenation of apolar substrates but the initial constructs are more susceptible to oxidative degradation than wild-type enzyme. Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.

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CcP(triAla)-catalyzed oxidation of 1-methoxynaphthalene by hydrogen peroxide. Spectra were collected every minute for one hour after addition of hydrogen peroxide. The large increase in absorbance between 450 and 800 nm is due to Russig’s blue formation. Inset – Increase in the absorbance at 610 nm as a function of time after hydrogen peroxide addition. Experimental Conditions: [CcP(triAla)] = 2.0 μM, [1-methoxynaphthalene] = 0.50 mM, [H2O2] = 1.00 mM, pH 7.0, 0.100 M ionic strength potassium phosphate buffer, 25°C.
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Figure 2: CcP(triAla)-catalyzed oxidation of 1-methoxynaphthalene by hydrogen peroxide. Spectra were collected every minute for one hour after addition of hydrogen peroxide. The large increase in absorbance between 450 and 800 nm is due to Russig’s blue formation. Inset – Increase in the absorbance at 610 nm as a function of time after hydrogen peroxide addition. Experimental Conditions: [CcP(triAla)] = 2.0 μM, [1-methoxynaphthalene] = 0.50 mM, [H2O2] = 1.00 mM, pH 7.0, 0.100 M ionic strength potassium phosphate buffer, 25°C.

Mentions: We have utilized the Russig’s blue assay developed by Shoji and coworkers [12] to assess the 1-methoxynaphthalene hydroxylation activity of CcP and the three CcP mutants with apolar distal heme pockets, CcP(triAla), CcP(triLeu), and CcP(triVal). Figure 2 shows the CcP(triAla) catalyzed oxidation of 1-methoxynaphthalene by hydrogen peroxide at pH 7.0. There is a large increase in the absorbance between 500 and 800 nm during the reaction that is characteristic of Russig’s blue formation resulting from the hydroxylation of 1-methoxynaphthalene, Figure 1[12]. The inset of Figure 2 shows the increase in the absorbance at 610 nm as a function of time from which the initial velocity can be determined, Equation 3. The initial velocity increases linearly with increasing CcP(triAla) concentration, Figure 3, from which a turnover number of 0.150 ± 0.008 min-1 can be determined, Table 1.


Peroxygenase activity of cytochrome c peroxidase and three apolar distal heme pocket mutants: hydroxylation of 1-methoxynaphthalene.

Erman JE, Kilheeney H, Bidwai AK, Ayala CE, Vitello LB - BMC Biochem. (2013)

CcP(triAla)-catalyzed oxidation of 1-methoxynaphthalene by hydrogen peroxide. Spectra were collected every minute for one hour after addition of hydrogen peroxide. The large increase in absorbance between 450 and 800 nm is due to Russig’s blue formation. Inset – Increase in the absorbance at 610 nm as a function of time after hydrogen peroxide addition. Experimental Conditions: [CcP(triAla)] = 2.0 μM, [1-methoxynaphthalene] = 0.50 mM, [H2O2] = 1.00 mM, pH 7.0, 0.100 M ionic strength potassium phosphate buffer, 25°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733812&req=5

Figure 2: CcP(triAla)-catalyzed oxidation of 1-methoxynaphthalene by hydrogen peroxide. Spectra were collected every minute for one hour after addition of hydrogen peroxide. The large increase in absorbance between 450 and 800 nm is due to Russig’s blue formation. Inset – Increase in the absorbance at 610 nm as a function of time after hydrogen peroxide addition. Experimental Conditions: [CcP(triAla)] = 2.0 μM, [1-methoxynaphthalene] = 0.50 mM, [H2O2] = 1.00 mM, pH 7.0, 0.100 M ionic strength potassium phosphate buffer, 25°C.
Mentions: We have utilized the Russig’s blue assay developed by Shoji and coworkers [12] to assess the 1-methoxynaphthalene hydroxylation activity of CcP and the three CcP mutants with apolar distal heme pockets, CcP(triAla), CcP(triLeu), and CcP(triVal). Figure 2 shows the CcP(triAla) catalyzed oxidation of 1-methoxynaphthalene by hydrogen peroxide at pH 7.0. There is a large increase in the absorbance between 500 and 800 nm during the reaction that is characteristic of Russig’s blue formation resulting from the hydroxylation of 1-methoxynaphthalene, Figure 1[12]. The inset of Figure 2 shows the increase in the absorbance at 610 nm as a function of time from which the initial velocity can be determined, Equation 3. The initial velocity increases linearly with increasing CcP(triAla) concentration, Figure 3, from which a turnover number of 0.150 ± 0.008 min-1 can be determined, Table 1.

Bottom Line: Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044 ± 0.0001 min-1.Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity.Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The cytochrome P450s are monooxygenases that insert oxygen functionalities into a wide variety of organic substrates with high selectivity. There is interest in developing efficient catalysts based on the "peroxide shunt" pathway in the cytochrome P450s, which uses H2O2 in place of O2/NADPH as the oxygenation agent. We report on our initial studies using cytochrome c peroxidase (CcP) as a platform to develop specific "peroxygenation" catalysts.

Results: The peroxygenase activity of CcP was investigated using 1-methoxynaphthalene as substrate. 1-Methoxynaphthalene hydroxylation was monitored using Russig's blue formation at standard reaction conditions of 0.50 mM 1-methoxynaphthalene, 1.00 mM H2O2, pH 7.0, 25°C. Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044 ± 0.0001 min-1. Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity. The highest activity was observed for CcP(triAla), a triple mutant with Arg48, Trp51, and His52 simultaneously mutated to alanine residues. The turnover number of CcP(triAla) is 0.150 ± 0.008 min-1, 34-fold greater than wild-type CcP and comparable to the naphthalene hydroxylation activity of rat liver microsomal cytochrome P450. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, CcP(triAla) is inactivated within four cycles of the peroxygenase reaction.

Conclusions: Protein engineering of CcP can increase the rate of peroxygenation of apolar substrates but the initial constructs are more susceptible to oxidative degradation than wild-type enzyme. Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.

Show MeSH
Related in: MedlinePlus