title: Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding.
abstract: Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.
authors.affiliation: Dip. Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Roma, Italy.
authors.forename: Antimo
authors.initials: A
authors.lastname: Cutone
authors.affiliation: Dip. Chimica 'Ugo Schiff', Università di Firenze, Sesto Fiorentino (FI), Italy.
authors.forename: Barry D
authors.initials: BD
authors.lastname: Howes
authors.affiliation: Dip. Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Roma, Italy.
authors.forename: Adriana E
authors.initials: AE
authors.lastname: Miele
authors.affiliation: Dip. Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Roma, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Miele
authors.affiliation: Dip. Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Roma, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Giorgi
authors.affiliation: Dip. Biologia, Università di Roma Tor Vergata, Roma, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Battistoni
authors.affiliation: Dip. Chimica 'Ugo Schiff', Università di Firenze, Sesto Fiorentino (FI), Italy.
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
authors.affiliation: Dip. Bioscienze e Territorio, Università del Molise, Pesche, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Musci
authors.affiliation: Dip. Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Roma, Italy.
authors.forename: Maria Carmela Bonaccorsi
authors.initials: MC
authors.lastname: di Patti