CONVERSION OF CYTOCHROME B(562) TO C-TYPE CYTOCHROMES

Cytochrome b(562) from the periplasm of Escherichia coli is the only m ember of a family of cytochromes sharing the 4-alpha-helical bundle st ructural motif that does not have a covalently bound heme. We have int roduced cysteine residues into the amino acid sequence of cytochrome b (562) in positions homologous to those found in the other members of t he family, generating the ubiquitous heme-binding peptide (-C-X-Y-C-H- ) found in virtually all c-type cytochromes. The resulting single-cyst eine-containing mutants, R98C and Y101C, together with the double muta nt combining both of these mutations have been expressed into the peri plasm of E. coli. The apo- and holoprotein products of each mutation h ave been isolated, and all the mutants produce multiple species with c ovalently attached heme. Results from ion exchange chromatography, opt ical spectroscopy, SDS gel electrophoresis, and electrospray mass spec trometry identified those species that appear to be cytochrome b(562) holoprotein with thioether covalent Linkages to the heme as the only d ifference in chemical composition between them and the wild-type prote in. Results from H-1-NMR experiments prove the existence of the expect ed c-type covalent bonds in each of these proteins and show that the s tructure of the heme pocket is not significantly perturbed by the cova lent modification(s). These proteins all have perturbed optical spectr a, compared with those of the wild-type protein, that are consistent w ith the modifications but are still characteristic of six-coordinate, low-spin cytochromes with Met-His ligation to the heme iron in both ox idation states.