Paramagnetic 13C and 15N NMR analyses of the push and pull effects in cytochrome c peroxidase and Coprinus cinereus peroxidase variants

Functional roles of highly conserved amino acids around heme

Daisuke Nonaka, Hiroyuki Wariishi, Karen G. Welinder, Hiroshi Fujii

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Paramagnetic 13C and 15N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide (13C15N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The 13C and 15N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The 13CNMRshifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.

Original languageEnglish
Pages (from-to)49-57
Number of pages9
JournalBiochemistry
Volume49
Issue number1
DOIs
Publication statusPublished - Jan 12 2010

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Coprinus
Cytochrome-c Peroxidase
Heme
Peroxidase
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Amino Acids
Hydrogen bonds
Hydrogen Bonding
Catalysis
Peroxidases
Mutation
Globins
Cyanides
Viperidae
Nuclear magnetic resonance spectroscopy
Hydrogen
Rate constants
Crystal structure
Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Paramagnetic 13C and 15N NMR analyses of the push and pull effects in cytochrome c peroxidase and Coprinus cinereus peroxidase variants: Functional roles of highly conserved amino acids around heme",
abstract = "Paramagnetic 13C and 15N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide (13C15N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The 13C and 15N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The 13CNMRshifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.",
author = "Daisuke Nonaka and Hiroyuki Wariishi and Welinder, {Karen G.} and Hiroshi Fujii",
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T1 - Paramagnetic 13C and 15N NMR analyses of the push and pull effects in cytochrome c peroxidase and Coprinus cinereus peroxidase variants

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AU - Nonaka, Daisuke

AU - Wariishi, Hiroyuki

AU - Welinder, Karen G.

AU - Fujii, Hiroshi

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N2 - Paramagnetic 13C and 15N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide (13C15N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The 13C and 15N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The 13CNMRshifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.

AB - Paramagnetic 13C and 15N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide (13C15N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The 13C and 15N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The 13CNMRshifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.

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