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J Biol Chem, Vol. 273, Issue 20, 12006-12016, May 15, 1998

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

J. Andrew Keightley^§, Donita Sanders, Thomas R. Todaro, Andrzej Pastuszyn^¶, and James A. Fee^§

From the  Department of Biology, University of California at San Diego, La Jolla, California 92093, the ^§ Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and the ^¶ Protein Chemistry Laboratory and Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, New Mexico 87131-5221

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 -band at 552 nm, contains ~60-70% of a protein highly similar to native cytochrome c₅₅₂ and ~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 -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 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.

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