Construction and characterization of a manganese-binding site in cytochrome c peroxidase: Towards a novel manganese peroxidase

Bryan K.S. Yeung, Xiaotang Wang, Jeffrey A. Sigman, Peter A. Petillo, Yi Lu

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III is a key step in the biodegradation of lignin,a complex phenylpropanoid polymer, as well as of many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. Results: Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41→Glu, Va145→Glu, His181→Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CcP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. Conclusions: A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.

Original languageEnglish (US)
Pages (from-to)215-221
Number of pages7
JournalChemistry and Biology
Volume4
Issue number3
DOIs
StatePublished - Mar 1997

Keywords

  • bioinorganic chemistry
  • metalloprotein
  • paramagnetic NMR
  • protein engineering
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Organic Chemistry

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