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.
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.
Keightley
J A
JA
Department of Biology, University of California at San Diego, La Jolla, California 92093, USA.
Sanders
D
D
Todaro
T R
TR
Pastuszyn
A
A
Fee
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology*
D003574:Cytochrome c Group; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C083951:cytochrome c553; C022302:cytochrome C-552