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

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• 作者：Manliang Feng ; Hiroyasu Tachikawa ; Xiaotang Wang ; Thomas D. Pfister ; Alan J. Gengenbach and Yi Lu
• 关键词：Cytochrome c peroxidase ; Manganese peroxidase ; Raman spectroscopy ; Site ; directed mutagenesis
• 刊名：Journal of Biological Inorganic Chemistry
• 出版年：2003
• 出版时间：September 2003
• 年：2003
• 卷：8
• 期：7
• 页码：699-706
• 全文大小：312 KB

文摘

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 ₃ 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).