Redox-Linked Structural Changes Associated with the Formation of a Catalytically Competent Form of the Diheme Cytochrome c Peroxidase from Pseudomonas aeruginosa

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• 作者：Aude Echalier ; Thomas Brittain ; Joshua Wright ; Svetlana Boycheva ; Gulnahar B. Mortuza ; Vilmos F&uuml ; lö ; p ; Nicholas J. Watmough
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：February 19, 2008
• 年：2008
• 卷：47
• 期：7
• 页码：1947 - 1956
• 全文大小：327K
• 年卷期：v.47,no.7(February 19, 2008)
• ISSN：1520-4995

文摘

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) fromPseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity.This material was used to carry out the first integrated biochemical, spectroscopic and structural investigationof the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca²⁺ ion(K = 3 × 10¹⁰ M^-1) found at the domain interface of both the fully oxidized and mixed-valence formsof the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca²⁺ ions triggers substantial structural rearrangements around theactive-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca²⁺ ions, but a combination of electronic absorption, and EPRspectroscopies suggests that under these circumstances the low potential heme remains six-coordinate,unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that thetwo mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N.,Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227-237) correspondto the Ca²⁺-loaded and -depleted forms of the enzyme.