RÉSUMÉ
The synthesis and characterization of short peptide-based maquettes of metalloprotein active sites facilitate an inquiry into their structure/function relationships and evolution. The [4Fe-4S]-maquettes of bacterial ferredoxin metalloproteins (Fd) have been used in the past to engineer redox active centers into artificial metalloenzymes. The novelty of our study is the application of maquettes to the superfamily of [4Fe-4S] cluster and S-adenosylmethionine-dependent radical metalloenzymes (radical SAM). The radical SAM superfamily enzymes contain site-differentiated, redox active [4Fe-4S] clusters coordinated to Cx3Cx2C or related motifs, which is in contrast to the Cx2Cx2C motif found in bacterial ferredoxins (Fd). Under an optimized set of experimental conditions, a high degree of reconstitution (80-100%) was achieved for both radical SAM- and Fd-maquettes. Negligible chemical speciation was observed for all sequences, with predominantly [4Fe-4S]2+ for the 'as-reconstituted' state. However, the reduction of [4Fe-4S]2+-maquettes provides low conversion (7-17%) to the paramagnetic [4Fe-4S]+ state, independent of either the spacing of the cysteine residues (Cx3Cx2C vs. Cx2Cx2C), the nature of intervening amino acids, or the length of the cluster binding motif. In the absence of the stabilizing protein environment, the reduction process is proposed to proceed via [4Fe-4S]2+ cluster disassembly and reassembly in a more reduced state. UV-Vis and EPR spectroscopic techniques are employed as analytical tools to quantitate the as-reconstituted (or oxidized) and one-electron reduced states of the [4Fe-4S] clusters, respectively. We demonstrate that short Fd and radical SAM derived 7- to 9-mer peptides containing appropriate cysteine motifs function equally well in coordinating redox active [4Fe-4S] clusters.