Cloning and Expression in Escherichia coli of the Cytochrome c552 Gene from Thermus thermophilus HB8

• Published in: The Journal of biological chemistry 1998-05, Vol.273 (20), p.12006-12016
• Main Authors: Keightley, J. Andrew, Sanders, Donita, Todaro, Thomas R., Pastuszyn, Andrzej, Fee, James A.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.20.12006

We report sequence of Thermus thermophilus HB8 DNA containing the gene (cycA) for cytochrome c552 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 rC552, characterized by an α-band at 552 nm, contains ∼60–70% of a protein highly similar to native cytochromec552 and ∼30–40% of a protein that contains a modified heme. Cytochrome rC552 is monomeric and is an excellent substrate for cytochromeba3. Cytochrome rC557is 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 cytochromeba3. We suggest thatrC557 is a “conformational isomer” ofrC552 having non-native, axial ligands to the heme iron and an “incorrect” protein fold that is stabilized by homodimer formation.