JBIC Journal of Biological Inorganic Chemistry

, Volume 8, Issue 7, pp 699–706 | Cite as

Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase

  • Manliang Feng
  • Hiroyasu Tachikawa
  • Xiaotang Wang
  • Thomas D. Pfister
  • Alan J. Gengenbach
  • Yi Lu
Original Article

Abstract

Cytochrome c peroxidase (CcP) variants with an engineered Mn(II) binding site, including MnCcP [CcP(MI, G41E, V45E, H181D)], MnCcP(W191F), and MnCcP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200–700 cm−1 and 1300–1650 cm−1 regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of CcP(MI), the recombinant yeast CcP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the ν3 mode indicate that a pure five-coordinate heme iron exists in CcP(MI) whereas a six-coordinate low-spin iron is the dominant species in the CcP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants CcP(MI, W191F) and CcP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H2O2 and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnCcP(W191F, W51F).

Keywords

Cytochrome c peroxidase Manganese peroxidase Raman spectroscopy Site-directed mutagenesis 

Abbreviations

CcP

cytochrome c peroxidase

CcP(MI)

recombinant yeast CcP containing Met-Ile at the N-terminus in addition to the normal wild-type CcP sequence

HRP

horseradish peroxidase

MnCcP

CcP(MI, G41E, V45E, H181D)

MnCcP(W191F)

CcP(MI, G41E, V45E, H181D, W191F)

MnCcP(W191F, W51F)

CcP(MI, G41E, V45E, H181D, W191F, W51F)

MnP

manganese peroxidase

RR

resonance Raman

WtCcP

wild-type cytochrome c peroxidase

Supplementary material

supp.pdf (82 kb)
Supplementary material (PDF 83 KB)

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Copyright information

© SBIC 2003

Authors and Affiliations

  • Manliang Feng
    • 1
  • Hiroyasu Tachikawa
    • 1
  • Xiaotang Wang
    • 1
  • Thomas D. Pfister
    • 2
  • Alan J. Gengenbach
    • 2
  • Yi Lu
    • 2
  1. 1.Department of ChemistryJackson State UniversityJacksonUSA
  2. 2.Departments of Chemistry and BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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