Mutational Analysis of the Cysteine-Rich Region of the Iron-Responsive GATA Factor Fep1. Role of Individual Cysteines as [2Fe-2S] Cluster Ligands.

Fep1, the iron-dependent GATA-type transcriptional repressor of the methylotrophic yeast Pichia pastoris, has a dimeric structure and binds an iron-sulfur cluster of the [2Fe-2S] type. In this work, we extend the characterization of this protein by analysis of the optical and CD spectroscopic properties of a set of mutants where cysteines within the conserved Cys-X5-Cys-X8-Cys-X2-Cys motif have been targeted, in order to evaluate their role as [2Fe-2S] ligands. The results suggest that all four cysteine residues are essential because replacing them with serines in different combinations invariably produces a protein unable to correctly bind the [2Fe-2S] cluster.