Role of calcium in maintaining the heme environment of manganese peroxidase

Greg R.J. Sutherland, Laura Schick Zapanta, Ming Tien, Steven D. Aust

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Abstract

We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland and Aust (1996) Arch. Blochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pK(a) of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near- ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

Original languageEnglish (US)
Pages (from-to)3654-3662
Number of pages9
JournalBiochemistry
Volume36
Issue number12
DOIs
StatePublished - Mar 25 1997

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manganese peroxidase
Heme
Calcium
Hot Temperature
Enzymes
Iron
Phanerochaete
Ligands
Hemeproteins
Electron Spin Resonance Spectroscopy
Arches
Circular Dichroism
Fungi
Histidine
Hydrogen Peroxide

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Sutherland, Greg R.J. ; Zapanta, Laura Schick ; Tien, Ming ; Aust, Steven D. / Role of calcium in maintaining the heme environment of manganese peroxidase. In: Biochemistry. 1997 ; Vol. 36, No. 12. pp. 3654-3662.
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Role of calcium in maintaining the heme environment of manganese peroxidase. / Sutherland, Greg R.J.; Zapanta, Laura Schick; Tien, Ming; Aust, Steven D.

In: Biochemistry, Vol. 36, No. 12, 25.03.1997, p. 3654-3662.

Research output: Contribution to journalArticle

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T1 - Role of calcium in maintaining the heme environment of manganese peroxidase

AU - Sutherland, Greg R.J.

AU - Zapanta, Laura Schick

AU - Tien, Ming

AU - Aust, Steven D.

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N2 - We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland and Aust (1996) Arch. Blochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pK(a) of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near- ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

AB - We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland and Aust (1996) Arch. Blochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pK(a) of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near- ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

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