Synthesis and characterization of singly modified (carboxyferrocenyl)cytochrome c derivatives

Carbodiimide-activated coupling chemistry has been used to covalently attach 1,1'-dicarboxyferrocene (dcFc) to the epsilon-amine of surface lysine residues of horse heart cytochrome c. Conditions have been found that optimize the production of singly modified (dcFc)cytochrome c derivatives and the presence of one free carboxylate per modification site allows separation and purification of about 10 of these derivatives by cation-exchange chromatography. Reversed-phase HPLC tryptic peptide mapping techniques have been used to identify the attachment sites of eight pure (dcFc)cytochrome c derivatives (at lysines 7, 8, 13, 25, 60, 72, 73, and 100). Through-space distances from these lysines to the nearest heme edge span the 6-16 A range and these derivatives should prove useful in exploring the distance dependence of long-range intramolecular electron transfer in cytochrome c.