Abstract
[Fe₄S₄]- and [Fe₃S₄]-clusters are ubiquitous iron-sulfur motifs in biological systems. The [Fe₃S₄] composition is, however, of much lower natural abundance than the more typical [Fe₄S₄]-clusters. In the present study formation of [Fe₃S₄]-clusters has been examined using chemically synthesized model peptides consisting of 33 amino acids (maquettes). Maquettes are effective synthetic analogs for metal-ion binding sites, allowing for a facile modification of the primary coordination sphere of iron-sulfur clusters. Maquettes have been designed following the [FeS]-cluster-binding motif of dimethyl sulfoxide reductase subunit B (DmsB) from Escherichia coli that carries a [Fe₄S₄]-cluster, but incorporates a [Fe₃S₄]-cluster instead upon mutation of one of the coordinating cysteines. The time-dependent formation of iron-sulfur clusters and the effects of exchanging selected amino acids in the model peptides, known to regulate the [Fe₃S₄] to [Fe₄S₄] ratio in the DmsB protein, were monitored by UV/Vis- and EPR-spectroscopy. Exchange of cysteines within the conserved CxxCxxC motif has a much stronger effect on cluster formation and stoichiometry than the exchange of a coordinating external cysteine. Amino acid exchange in the binding motif shows a dependence of the cluster stoichiometry on the amino acid side chain. Formation of [Fe₃S₄]-clusters in maquettes is less favorable compared to native proteins. The [Fe₃S₄] moiety appears to be a rather transient species towards the more stable (final) incorporation of a [Fe₄S₄]-cluster. Results are best described by an assembly mechanism that considers a successive coordination of the iron atoms by the peptide, rather than incorporation of an already pre-formed mercaptoethanol-coordinated [Fe₄S₄]-cluster.