2ICV

Kinetic and Crystallographic Studies of a Redesigned Manganese-Binding Site in Cytochrome c Peroxidase

2ICV の概要

• エントリーDOI: 10.2210/pdb2icv/pdb
• 分子名称: Cytochrome c peroxidase, mitochondrial, COBALT (II) ION, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• 機能のキーワード: manganese oxidation, metal-binding site, protein engineering, metalloprotein, biomimetic, oxidoreductase
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• 細胞内の位置: Mitochondrion matrix: P00431
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 33936.33

構造登録者

Pfister, T.,Mirarefi, A.Y.,Gengenbach, A.J.,Zhao, X.,Conaster, C.D.N.,Gao, Y.G.,Robinson, H.,Zukoski, C.F.,Wang, A.H.J.,Lu, Y. (登録日: 2006-09-13, 公開日: 2007-08-28, 最終更新日: 2023-08-30)

主引用文献

Pfister, T.D.,Mirarefi, A.Y.,Gengenbach, A.J.,Zhao, X.,Danstrom, C.,Conatser, N.,Gao, Y.-G.,Robinson, H.,Zukoski, C.F.,Wang, A.H.-J.,Lu, Y.
Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase
J.Biol.Inorg.Chem., 12:126-137, 2007

PubMed Abstract: Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.

PubMed: 17021923
DOI: 10.1007/s00775-006-0171-0

実験手法

X-RAY DIFFRACTION (1.6 Å)