Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10843490 | Protein Expression and Purification | 2005 | 9 Pages |
Abstract
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 CuA-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000Â nmol, equivalent to 65Â mg, of the CuA-cytochrome c protein per litre of Escherichia coli culture. About 500Â 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 CuA site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the CuA-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and CuA sub-domains behave independently despite their close physical and functional association.
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Authors
Diann Andrews, Neil R. Mattatall, Danielle Arnold, Bruce C. Hill,