Greiner Peter, Hannappel Achim, Werner Carolin, Ludwig Bernd
Institute for Biochemistry, Molecular Genetics, Johann-Wolfgang-Goethe University, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):904-11. doi: 10.1016/j.bbabio.2008.04.003. Epub 2008 Apr 10.
Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of cytochrome c oxidase using the soil bacterium Paracoccus denitrificans as a model organism. The use of bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is involved in copper delivery to the Cu(B) center, has been purified and characterized spectroscopically. A previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of "solubilized" subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes.
细胞色素c氧化酶的生物合成是一个复杂的过程，在酵母中涉及30多种已知的辅助蛋白，用于转录和翻译的调控、膜插入和蛋白质加工、辅因子插入以及亚基组装。在这里，我们以土壤细菌反硝化副球菌作为模式生物，重点研究细胞色素c氧化酶亚基I中辅因子的插入过程。使用细菌系统有助于生物合成研究，因为所需组装因子的数量被减少到最低限度。本文讨论了共翻译和翻译后辅因子插入的情况，并提出了几种阐明生物合成这一方面的方法。已对参与向Cu(B)中心输送铜的酵母Cox11的反硝化副球菌同源物CtaG进行了纯化和光谱表征。在358 nm处一个以前未报道的信号可以监测铜从负载铜的CtaG转移到受体。在大肠杆菌中表达后，纯化了CtaG和脱辅基亚基I，以建立体外铜转移系统，探究翻译后插入假说。为了模拟潜在的共翻译插入过程，已经建立了使用大肠杆菌和反硝化副球菌提取物的无细胞表达系统。在去污剂Brij-35存在的情况下表达亚基I会产生大量“可溶”的亚基I，其可以以良好的产率纯化。使用该系统，在添加游离辅因子、纯化的组装蛋白或反硝化副球菌膜后，捕获和纯化组装中间体可能是可行的。
