Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis

Bacillus subtilis expresses a cytochrome c-550nm that participates in respi ratory electron transfer and is an integral membrane protein. Analysis of t he B. subtilis cytochrome c-550nm amino acid sequence predicts a single N-t erminal transmembrane helix attached to a water-soluble heme binding domain [C. von Wachenfeldt and L. Hederstedt (1990) J. Biol. Chem, 265, 13939-139 48]. We have purified cytochrome c-550nm from wild-type B. subtilis and B. subtilis transformed with the shuttle vector pHP13 containing the gene for B. subtilis cytochrome c-550nm (cccA), In B, subtilis transformed with pHP1 3/cccA there is better than eightfold more membrane-bound cytochrome c-550n m than in wildtype B, subtilis, The overexpressed cytochrome c-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media. A six -histidine tag has been added to the C-terminus of cytochrome c-550nm from B. subtilis as a further aid for purification. This strain produces cytochr ome c-550nm to a level fourfold greater than wild type and allows for one-s tep purification using metal affinity chromatography, UV-Vis spectroscopy d etects no change in the heme C spectrum due to the addition of six histidin es, Neither form of B. subtilis cytochrome c-550nm is stable in its reduced state in aerated buffer, unless EDTA is added. The two forms, wild-type an d his-tagged, of cytochromes c have similar midpoint redox potentials of 19 5 and 185 mV,respectively, and are equally good substrates for B. subtilis cytochrome c oxidase, We conclude that the addition of the histidine tag ea ses the purification of cytochrome c-550nm from B. subtilis plasma membrane s and that the additional metal binding site does not compromise the stabil ity or functional properties of the protein. (C) 1999 Academic Press.