1CYF

IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING

1CYF の概要

• エントリーDOI: 10.2210/pdb1cyf/pdb
• 関連するPDBエントリー: 1CCP
• 分子名称: CYTOCHROME C PEROXIDASE, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• 機能のキーワード: oxidoreductase (h2o2(a))
• 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: 34386.09

構造登録者

Miller, M.A.,Han, G.W.,Kraut, J. (登録日: 1995-07-03, 公開日: 1995-12-07, 最終更新日: 2024-02-07)

主引用文献

Miller, M.A.,Geren, L.,Han, G.W.,Saunders, A.,Beasley, J.,Pielak, G.J.,Durham, B.,Millett, F.,Kraut, J.
Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering.
Biochemistry, 35:667-673, 1996

PubMed Abstract: A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)-biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET.

PubMed: 8547245
DOI: 10.1021/bi952557a

実験手法

X-RAY DIFFRACTION (2.35 Å)