Cell Biochemistry and Biophysics https://doi.org/10.1007/s12013-018-0842-9 ORIGINAL PAPER Mutational Analysis of the Cysteine-Rich Region of the Iron- Responsive GATA Factor Fep1. Role of Individual Cysteines as [2Fe–2S] Cluster Ligands Maria Carmela Bonaccorsi di Patti 1 ● Antimo Cutone 2 ● Giovanni Musci 2 Received: 26 January 2018 / Accepted: 30 March 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018 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-X 5 - Cys-X 8- Cys-X 2 -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. Keywords: Iron ● Iron–sulfur ● Yeast ● Fep1 Introduction Regulation of intracellular levels of iron is critical for living beings ranging from bacteria to humans and cells have been found to employ different modes of regulation of gene expression to control uptake and distribution of this metal. Generally, in fungi and bacteria iron-responsive transcrip- tion factors cooperate to maintain adequate levels of iron in order to respond to iron starvation or excess through com- plex regulatory networks that are beginning to be unveiled [1, 2]. In a large number of fungi, transcriptional repressors sensitive to iron have been identified, which are called GATA factors as they interact and bind to a 5′-(A/T) GATAA-3′ sequence on DNA. Previously, we demon- strated that Fep1, one of these transcriptional factors, con- trols entry of iron in the methylotrophic yeast Pichia pastoris [3, 4], confirming that GATA factors share a common role in iron homeostasis regulation. As far as the structure is concerned, the GATA factors of fungi are composed of two functionally distinct domains, with the N-terminal domain involved in binding to DNA and the C-terminal one managing the repression function. Two zinc fingers of the Cys 2 –Cys 2 type (hereafter referred to as ZnF1 and ZnF2) are present in the N-terminal domain, separated by a sequence which invariably contains a set of four cysteines within a Cys-X 5 -Cys-X 8 -Cys-X 2 -Cys motif (the “central” cysteines). The primary structure of the C- terminal domain, on the other hand, is not conserved among different iron-sensitive GATA factors. This domain is suggested to play a role in dimerization and interaction with other protein partners [1]. The Cys-X 5 -Cys-X 8 -Cys-X 2 -Cys motif is strictly required for fungal GATA factors to be able to respond to iron levels and binding of iron has been established for Neurospora crassa Sre [5], Histoplasma capsulatum Sre1 [6] and Schizosaccharomyces pombe Fep1 [7, 8]. We have demonstrated by SAXS, optical and resonance Raman spectroscopies that P. pastoris Fep1 exists as a dimer and binds iron in the form of a [2Fe–2S] cluster in anaerobic and aerobic conditions [4]. In this work, we aimed at specifically evaluating the role of each residue of the Cys-X 5 -Cys-X 8 -Cys-X 2 -Cys motif as [2Fe–2S] ligand in P. pastoris Fep1. To this purpose, we extended the characterization of the protein by analysis of the optical and circular dichroism (CD) spectroscopic * Giovanni Musci musci@unimol.it 1 Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy 2 Department of Biosciences and Territory, University of Molise, Pesche, Italy 1234567890();,: 1234567890();,: