Synthetic mutants of Clostridium pasteurianum ferredoxin: open iron sites and testing carboxylate coordination

The entire polypeptide chains for two new Clostridium pasteurianumferredoxin (Fd) mutants were prepared with the following site-specificsubstitutions: 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 twoapoproteins were reconstituted to the 2[4Fe-4S] holoprotein and theirspectroscopic, redox and thermal properties were compared with those ofnative C.pasteurianum Fds. The fully reconstituted Cys11Asp and Cys11alpha-Aba mutants were initially found to have both clusters intact, i.e.they were 2[4Fe-4S] ferredoxins. The unconventional ligands of Asp andalpha-Aba led to holo-Fds that were not very stable and easily released aniron to form the [3Fe-4S] cluster, presumably through oxidation. The Cys11alpha-Aba mutant was somewhat more thermally stable than Cys11Asp. Incontrast, while both mutants were less stable than the native protein uponexposure to oxygen, the Cys11 alpha-Aba mutant was less stable thanCys11Asp. The Cys11Gly mutant was also prepared, but all attempts, despiterepeated and varied experimental conditions, at reconstitution to theCys11Gly holo 2[4Fe-4S] Fd were unsuccessful, probably because a Gly-Glysequence is known to break structure. This work, when compared withmolecular biological site- specific mutagenesis, shows some of theadvantages of chemical/in vitro reconstitution: certain mutants whichcannot be detected as holoproteins by site-specific mutagenesis can beformed after all in vitro. Nonetheless, it seems apparent that altering anyof the Cys coordination sites of the Fd clusters results in fundamentallymore unstable ferredoxins.