Cenacchi Lucia, Busch Manuela, Schleidt Philipp G, Müller Florian G, Stumpp Tina V M, Mäntele Werner, Trost Paolo, Lancaster C Roy D
Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Frankfurt, Germany.
Biochim Biophys Acta. 2012 Mar;1818(3):679-88. doi: 10.1016/j.bbamem.2011.10.030. Epub 2011 Nov 7.
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 Fe(3+)-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 Fe(3+)-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.
细胞色素（cyt）b(561)蛋白是含两个血红素的膜蛋白，属于CYBASC（细胞色素b(561)-抗坏血酸可还原）家族，据推测参与抗坏血酸循环和/或促进铁吸收。在此，我们展示了拟南芥的两个细胞色素b(561)旁系同源物（Acytb(561)-A、Acytb(561)-B）在大肠杆菌和巴斯德毕赤酵母中的异源表达、纯化及初步表征。光谱表明，Acytb(561)-A类似于CYBASC家族中特征最明确的成员——肾上腺髓质嗜铬小泡中的细胞色素b(561)，而Acytb(561)-B与其他CYBASC蛋白相比具有非典型性。发现血红素氧化还原中点电位（E(M)）值与抗坏血酸氧化活性和Fe(3+) -螯合物还原酶活性完全一致。如嗜铬小泡中的细胞色素b(561)报道的那样，发现两个旁系同源物的抗坏血酸依赖性还原和蛋白质稳定性对碱性pH值敏感。对于两个旁系同源物，在无抗坏血酸的情况下用焦碳酸二乙酯（DEPC）孵育会抑制抗坏血酸依赖性还原，并且低电位血红素E(M)值会受到显著影响。在有抗坏血酸存在的情况下用DEPC修饰，与未修饰的蛋白质相比，血红素E(M)值未改变。然而，抗坏血酸还原受到抑制。我们得出结论，抗坏血酸结合位点位于低电位血红素附近，Fe(3+) -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不仅通过降低血红素E(M)值，还通过影响抗坏血酸结合和/或电子转移的额外修饰。分析凝胶过滤实验表明，两个细胞色素b(561)旁系同源物均以同型二聚体形式存在。
