The entire polypeptide chains for two new Clostridium pasteurianum ferredoxin (Fd) mutants were prepared with the following site-specific substitutions: Cys11Asp and Cys11 alpha-aminobutyric acid (Cys11 alpha-Aba), the latter being a non-naturally occurring amino acid. Standard t-Boc procedures were used for the synthesis and the peptides. The two apoproteins were reconstituted to the 2[4Fe-4S] holoprotein and their spectroscopic, redox and thermal properties were compared with those of native C.pasteurianum Fds. The fully reconstituted Cys11Asp and Cys11 alpha-Aba mutants were initially found to have both clusters intact, i.e. they were 2[4Fe-4S] ferredoxins. The unconventional ligands of Asp and alpha-Aba led to holo-Fds that were not very stable and easily released an iron to form the [3Fe-4S] cluster, presumably through oxidation. The Cys11 alpha-Aba mutant was somewhat more thermally stable than Cys11Asp. In contrast, while both mutants were less stable than the native protein upon exposure to oxygen, the Cys11 alpha-Aba mutant was less stable than Cys11Asp. The Cys11Gly mutant was also prepared, but all attempts, despite repeated and varied experimental conditions, at reconstitution to the Cys11Gly holo 2[4Fe-4S] Fd were unsuccessful, probably because a Gly-Gly sequence is known to break structure. This work, when compared with molecular biological site-specific mutagenesis, shows some of the advantages of chemical/in vitro reconstitution: certain mutants which cannot be detected as holoproteins by site-specific mutagenesis can be formed after all in vitro. Nonetheless, it seems apparent that altering any of the Cys coordination sites of the Fd clusters results in fundamentally more unstable ferredoxins.