Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium

Ross E. Whitwam, Rao S. Koduri, Michael Natan, Ming Tien

Research output: Contribution to journalArticle

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Abstract

Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H2O2 were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn2+ from 233 s-1 (wild type) to 154 s-1 and 107 s-1, respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn2+ to 320 s-1. The F190L mutation decreased this rate to 137 s-1. The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn2+ from 140 s-1 in the wild type to 36 s-1. There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe3+/Fe2+ couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heine and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.

Original languageEnglish (US)
Pages (from-to)9608-9616
Number of pages9
JournalBiochemistry
Volume38
Issue number30
DOIs
StatePublished - Jul 27 1999

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manganese peroxidase
Phanerochaete
Ligands
Mutation
Peroxidases
Mutation Rate
Hydrogen
Rate constants
Enzymes
Mutagenesis
Hydrogen Bonding
Viperidae
Site-Directed Mutagenesis
Fungi
Heme
Oligonucleotides
Cations
Hydrogen bonds
Proteins
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Whitwam, Ross E. ; Koduri, Rao S. ; Natan, Michael ; Tien, Ming. / Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium. In: Biochemistry. 1999 ; Vol. 38, No. 30. pp. 9608-9616.
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abstract = "Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H2O2 were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn2+ from 233 s-1 (wild type) to 154 s-1 and 107 s-1, respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn2+ to 320 s-1. The F190L mutation decreased this rate to 137 s-1. The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn2+ from 140 s-1 in the wild type to 36 s-1. There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe3+/Fe2+ couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heine and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.",
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Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium. / Whitwam, Ross E.; Koduri, Rao S.; Natan, Michael; Tien, Ming.

In: Biochemistry, Vol. 38, No. 30, 27.07.1999, p. 9608-9616.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium

AU - Whitwam, Ross E.

AU - Koduri, Rao S.

AU - Natan, Michael

AU - Tien, Ming

PY - 1999/7/27

Y1 - 1999/7/27

N2 - Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H2O2 were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn2+ from 233 s-1 (wild type) to 154 s-1 and 107 s-1, respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn2+ to 320 s-1. The F190L mutation decreased this rate to 137 s-1. The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn2+ from 140 s-1 in the wild type to 36 s-1. There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe3+/Fe2+ couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heine and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.

AB - Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H2O2 were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn2+ from 233 s-1 (wild type) to 154 s-1 and 107 s-1, respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn2+ to 320 s-1. The F190L mutation decreased this rate to 137 s-1. The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn2+ from 140 s-1 in the wild type to 36 s-1. There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe3+/Fe2+ couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heine and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.

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