Redox-linked structural changes associated with the formation of a catalytically competent form of the diheme cytochrome c peroxidase from Pseudomonas aeruginosa
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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DOI：
10.1021/bi702064f
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发表时间：
2008-02-19
期刊：
影响因子：
2.9
通讯作者：
Watmough, Nicholas J.
中科院分区：
文献类型：
--
作者：
Echalier, Aude;Brittain, Thomas;Watmough, Nicholas J.
铜绿假单胞菌（Pseudomonas aeruginosa）的原型双血红素细菌细胞色素c过氧化物酶（BCCP）的重组形式（PsaCCP）已在大肠杆菌中表达并纯化至均一。该材料被用于对导致这类酶还原激活的因素进行首次综合的生化、光谱和结构研究。在该酶完全氧化态和混合价态的结构域界面处发现的单个紧密结合的Ca²⁺离子（K = 3×10¹⁰ M⁻¹）对催化活性是绝对必需的。在Ca²⁺离子存在下，电子传递（高电位）血红素的还原引发活性位点（低电位）血红素周围的大量结构重排，从而允许底物结合和催化。在没有Ca²⁺离子的情况下，该酶也会形成混合价态，但电子吸收和电子顺磁共振光谱的结合表明，在这些情况下，低电位血红素保持六配位，无法结合底物，因此无催化活性。我们的观察结果有力地表明，Foote及其同事先前报道的天然PsaCCP的两种混合价态（Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227 - 237）分别对应于该酶结合Ca²⁺和缺失Ca²⁺的形式。
A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas 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 investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of 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 the active-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 EPR spectroscopies 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 the two 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) correspond to the Ca2+-loaded and -depleted forms of the enzyme.