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.
Mutational Analysis of the Cysteine-Rich Region of the Iron-Responsive GATA Factor Fep1. Role of Individual Cysteines as [2Fe–2S] Cluster Ligands
Patti, Maria Carmela Bonaccorsi di;Cutone, Antimo;Musci, Giovanni
2018-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
