Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells
Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells
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DOI：
10.1016/j.bbamem.2011.10.030
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发表时间：
2012-03-01
影响因子：
3.4
通讯作者：
Lancaster, C. Roy D.
中科院分区：
文献类型：
--
作者：
Cenacchi, Lucia;Busch, Manuela;Lancaster, C. Roy D.
细胞色素（cyt） b（561）蛋白是一种含双血红蛋白的膜蛋白，属于CYBASC（细胞色素-b(561)-抗坏血酸可还原）家族，被认为参与抗坏血酸的回收和/或促进铁的吸收。在这里，我们介绍了拟南芥中两个cytb（561）类似物(Acytb（561)-A, Acytb(561)-B）在大肠杆菌和毕赤酵母中的异源生产，它们的纯化和初步表征。光谱显示，Acytb(561)-A类似于CYBASC家族中最具特征的成员，来自肾上腺髓质染色质囊泡的细胞色素b(561)，与其他CYBASC蛋白相比，Acytb(561)-B是不典型的。血红素氧化还原中点电位（E-M）值与抗坏血酸氧化活性和Fe3+螯合物还原酶活性完全一致。据报道，从染色质囊泡中提取的细胞色素b（561）的抗坏血酸依赖性减少和蛋白质稳定性对碱性pH值敏感。在没有抗坏血酸的情况下，用焦碳酸二乙酯（DEPC）孵育，抑制了抗坏血酸依赖的还原，并且显著影响了低电位血红素E-M值。与未修饰的蛋白质相比，在抗坏血酸存在的情况下用DEPC修饰血红素E-M值不变。然而，抗坏血酸的还原被抑制。我们得出结论，抗坏血酸结合位点位于低电位血红素附近，Fe3+螯合物还原位点靠近高电位血红素。此外，DEPC处理对抗坏血酸氧化的抑制作用不仅通过降低血红素E-M值发生，而且还通过影响抗坏血酸结合和/或电子转移的附加修饰发生。分析凝胶过滤实验表明，这两种cyt b（561）相似物以同型二聚体的形式存在。(C) 2011 Elsevier B.V.版权所有
Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E-M) values were found to be fully consistent with ascorbate oxidation activities and Fe3+-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E-M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E-M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe3+-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E-M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers. (C) 2011 Elsevier B.V. All rights reserved.