Andrews Diann, Mattatall Neil R, Arnold Danielle, Hill Bruce C
Department of Biochemistry, Queen's University, Kingston, Ont., Canada K7L 3N6.
Protein Expr Purif. 2005 Aug;42(2):227-35. doi: 10.1016/j.pep.2004.11.009. Epub 2004 Dec 16.
Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.
来自枯草芽孢杆菌的细胞色素caa3是血红素-铜氧化酶家族的一员，该家族属于整合膜酶，其中包括线粒体细胞色素c氧化酶。相对于线粒体中的对应物，细胞色素caa3的亚基II在其C末端有额外的100个氨基酸，并且该延伸区域编码一个血红素C结合结构域。细胞色素caa3具有线粒体细胞色素c与线粒体细胞色素c氧化酶之间形成的复合物的许多特性。为了更仔细地研究细胞色素c与氧化酶之间的相互作用，我们克隆并表达了来自枯草芽孢杆菌细胞色素caa3复合物的亚基II的Cu(A)-细胞色素c部分。每升大肠杆菌培养物中，我们能够表达约2000 nmol（相当于65 mg）的Cu(A)-细胞色素c蛋白。约500 nmol正确靶向至周质空间，并且我们通过亲和色谱和硫酸铵分级分离相结合的方法纯化了其中的50%。含有细胞色素c的亚结构域折叠良好，血红素C中心周围环境稳定，因为其中点电位和还原速率与全酶的细胞色素c结构域的值没有区别。然而，Cu(A)位点缺乏铜，导致该亚结构域存在内在不稳定性。以Cu(A)-细胞色素c蛋白为例，枯草芽孢杆菌细胞色素c可以在大肠杆菌中表达，并且我们得出结论：尽管细胞色素c和Cu(A)亚结构域在物理和功能上紧密相关，但它们的行为是独立的。
