Heme Is Not Required for<i>Aquifex aeolicus</i>Cytochrome<i>c</i>₅₅₅Polypeptide Folding

Abstract

In cytochrome c, it has been supposed that heme must bind to the apo polypeptide for structure formation. We constructed a C12A/C15A variant of hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) in which the covalently heme-binding Cys residues were replaced by Ala, and characterized its molecular features. The apo C12A/C15A variant had almost the same helical content as holo AA c(555), and spontaneously incorporated heme in vitro with no helical content change. These results suggest that the apo AA c(555) polypeptide is intrinsically structured without heme binding, this being the first case of a cytochrome c polypeptide. This finding provides a new suggestion as to cytochrome c formation, that heme is not necessarily required for cytochrome c polypeptide folding.