Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa.: The role of a heme-linked protein loop:: A mutagenesis studies
Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa.: The role of a heme-linked protein loop:: A mutagenesis studies
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DOI：
10.1016/j.jinorgbio.2007.04.012
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发表时间：
2007-08-01
影响因子：
3.9
通讯作者：
Brittain, Thomas
中科院分区：
文献类型：
--
作者：
Hsiao, Hsi-Chen;Boycheva, Svetlana;Brittain, Thomas
诱变研究已用于研究含血红素配体的蛋白质环（67-79）在二血红素过氧化物酶活化中的作用。铜绿假单胞菌的细胞色素c过氧化物酶的两种突变形式已经产生。一种突变体（环突变体）缺乏蛋白质环，另一种（H71 G）在正常组氨酸配体位点含有非连接性Gly。光谱数据表明，在这两个突变体的远端组氨酸配体的过氧化物血红素在未活化的酶丢失或可交换的。在过氧化氢和生理电子供体细胞色素c（551）存在下，未活化的H71 G和环突变体分别显示出活化形式的野生型酶的75%和10%的周转活性。这两种突变体蛋白质都表现出与过氧化物在通常无活性、完全氧化的酶形式下的组成性反应性，并产生自由基中间体。自由基产品的组成性过氧化物反应似乎是位于不同的网站在两个突变蛋白。这些结果表明，从过氧化物血红素的组氨酸配体的损失，本身就足以产生过氧化物活性，通过提供一个过氧化物结合位点和自由基中间体的形成是非常敏感的蛋白质结构的变化。总的来说，这些数据是一致的蛋白质环67-79在激活二血红素过氧化物酶的主要作用，并建议“电荷跳跃”机制可能是在分子内电子转移的过程中起作用。(c)2007年爱思唯尔公司All rights reserved.
Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71 G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c(551). Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a "charge hopping" mechanism may be operative in the process of intra-molecular electron transfer. (c) 2007 Elsevier Inc. All rights reserved.