Cloning and Expression in Escherichia coli of the Cytochrome c 552 Gene from Thermus thermophilus HB8

We report sequence of Thermus thermophilus HB8 DNA containing the gene ( cycA ) for cytochrome c 552 and a gene ( cycB ) encoding a protein homologous with one subunit of an ATP-binding cassette transporter. The cycA gene encodes a 17-residue N-terminal signal peptide with following amino acid sequence identical to that reported by (Titani, K., Ericsson, L. H., Hon-nami, K., and Miyazawa, T. (1985) Biochem. Biophys. Res. Commun. 128, 781–787). A modified cycA was placed under control of the T7 promoter and expressed in Escherichia coli. Protein identical to that predicted from the gene sequence was found in two heme C-containing fractions. Fraction rC 552, characterized by an α-band at 552 nm, contains ∼60–70% of a protein highly similar to native cytochrome c 552 and ∼30–40% of a protein that contains a modified heme. Cytochrome rC 552 is monomeric and is an excellent substrate for cytochrome ba 3. Cytochrome rC 557 is characterized by an α-band at 557 nm, contains ∼90% heme C and ∼10% of non-C heme, exists primarily as a homodimer, and is essentially inactive as a substrate for cytochrome ba 3. We suggest that rC 557 is a “conformational isomer” of rC 552 having non-native, axial ligands to the heme iron and an “incorrect” protein fold that is stabilized by homodimer formation.