In vitro formation of a c-type cytochrome

C-type cytochromes are essential for almost all organisms; they are characterized by the covalent attachment of heme to protein through two thioether bonds to a Cys-Xaa-Xaa-Cys-His peptide motif. Here we show, contrary to opinion of 30 years standing, that a c-type cytochrome can form from heme and apoprotein in vitro under mild conditions and in the absence of any biosynthesis apparatus. This reaction occurs provided formation of a disulfide bond within the Cys-Xaa-Xaa-Cys-His motif is avoided. There are important implications for understanding in vivo cytochrome c assembly.