Recombinant cytochrome rC(557) obtained from Escherichia coli cells expressing a truncated Thermus thermophilus cycA gene - Heme inversion in an improperly matured protein

Cytochrome rC(557) is an improperly matured, dimeric cytochrome c obtained from expression of the "signal peptide-lacking" Thermus thermophilus cycA g ene in the cytoplasm of Escherichia coli. It is characterized by its Q(00) (or alpha-) optical absorption band at 557 nm in the reduced form (Keightle y, J. A., Sanders, D., Todaro, T. R., Pastuszyn, A., and Fee, J. A. (1998) J. Biol. Chem. 273, 12006-12016). We report results of a broad ranging, bio chemical and spectral characterization of this protein that reveals the pre sence of a free vinyl group on the porphyrin and a disulfide bond between t he protomers and supports His-Met ligation in both valence states of the ir on. A 3-Angstrom resolution x-ray structure shows that, in comparison with the native protein, the heme moiety is rotated 180 degrees about its alpha, gamma -axis; cysteine 14 has formed a thioether bond with the 2-vinyl of py rrole ring I instead of the 4-vinyl of pyrrole ring II, as occurs in the na tive protein; and a cysteine 11 from each protomer has formed an intermolec ular disulfide bond. Numerous, minor perturbations exist within the structu re of rC(557) in comparison with that of native protein, which result from heme inversion and protein-protein interactions across the dimer interface. The unusual spectral properties of rC(557) are rationalized in terms of th is structure.