Cloning and expression in Escherichia coli of the cytochrome c552 gene from Thermus thermophilus HB8. Evidence for genetic linkage to an ATP-binding cassette protein and initial characterization of the cycA gene products

• Published in: The Journal of biological chemistry 1998-05, Vol.273 (20), p.12006-12016
• Main Authors: Keightley, J A, Sanders, D, Todaro, T R, Pastuszyn, A, Fee, J A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Chromatography, Gel; Cloning, Molecular; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Cytochrome c Group - isolation & purification; Dimerization; DNA, Bacterial; Escherichia coli - genetics; Mass Spectrometry; Molecular Sequence Data; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Sequence Homology, Amino Acid; Thermus thermophilus - enzymology
• ISSN: 0021-9258
• 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 alpha-band at 552 nm, contains approximately 60-70% of a protein highly similar to native cytochrome c552 and approximately 30-40% of a protein that contains a modified heme. Cytochrome rC552 is monomeric and is an excellent substrate for cytochrome ba3. Cytochrome rC557 is characterized by an alpha-band at 557 nm, contains approximately 90% heme C and approximately 10% of non-C heme, exists primarily as a homodimer, and is essentially inactive as a substrate for cytochrome ba3. We suggest that rC557 is a "conformational isomer" of rC552 having non-native, axial ligands to the heme iron and an "incorrect" protein fold that is stabilized by homodimer formation.