Heme Is Not Required for Aquifex aeolicus Cytochrome c₅₅₅ Polypeptide 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 c₅₅₅ (AA c₅₅₅) 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 c₅₅₅, and spontaneously incorporated heme in vitro with no helical content change. These results suggest that the apo AA c₅₅₅ 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.