Stability and Folding Kinetics of Structurally Characterized Cytochrome c-b_(562)

The four-helix-bundle protein fold can be constructed from a wide variety of primary amino acid sequences. Proteins with this structure are excellent candidates for investigations of the relationship between folding mechanism and topology. The folding of cytochrome b_(562), a four-helix-bundle heme protein, is hampered by heme dissociation. To overcome this complication, we have engineered a variant of cytochrome b_(562) (cyt c-b_(562)) featuring a c-type linkage between the heme and the polypeptide chain. The replacement of the native cyt b_(562) leader sequence in this protein with that of a c-type cytochrome (cyt c_(556)) led to high yields of fully matured and correctly folded cyt c-b_(562). We have determined the X-ray crystal structure of cyt c-b_(562) at 2.25 Å and characterized its physical, chemical, and folding properties. These measurements reveal that the c-type linkage does not perturb the protein fold or reduction potential of the heme group. The covalent attachment of the porphyrin to the polypeptide does, however, produce a substantial change in protein stability and folding kinetics.