title: Formation of [4Fe-4S] clusters in the mitochondrial iron-sulfur cluster assembly machinery.
abstract: The generation of [4Fe-4S] clusters in mitochondria critically depends, in both yeast and human cells, on two A-type ISC proteins (in mammals named ISCA1 and ISCA2), which perform a nonredundant functional role forming in vivo a heterocomplex. The molecular function of ISCA1 and ISCA2 proteins, i.e., how these proteins help in generating [4Fe-4S] clusters, is still unknown. In this work we have structurally characterized the Fe/S cluster binding properties of human ISCA2 and investigated in vitro whether and how a [4Fe-4S] cluster is assembled when human ISCA1 and ISCA2 interact with the physiological [2Fe-2S](2+) cluster-donor human GRX5. We found that (i) ISCA2 binds either [2Fe-2S] or [4Fe-4S] cluster in a dimeric state, and (ii) two molecules of [2Fe-2S](2+) GRX5 donate their cluster to a heterodimeric ISCA1/ISCA2 complex. This complex acts as an "assembler" of [4Fe-4S] clusters; i.e., the two GRX5-donated [2Fe-2S](2+) clusters generate a [4Fe-4S](2+) cluster. The formation of the same [4Fe-4S](2+) cluster-bound heterodimeric species is also observed by having first one [2Fe-2S](2+) cluster transferred from GRX5 to each individual ISCA1 and ISCA2 proteins to form [2Fe-2S](2+) ISCA2 and [2Fe-2S](2+) ISCA1, and then mixing them together. These findings imply that such heterodimeric complex is the functional unit in mitochondria receiving [2Fe-2S] clusters from hGRX5 and assembling [4Fe-4S] clusters before their transfer to the final target apo proteins.
authors.affiliation: Department of Pharmacy, University of Naples "Federico II" , Via D. Montesano 49, 80131 Napoli, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: Brancaccio
authors.forename: Angelo
authors.initials: A
authors.lastname: Gallo
authors.forename: Maciej
authors.initials: M
authors.lastname: Mikolajczyk
authors.forename: Kairit
authors.initials: K
authors.lastname: Zovo
authors.forename: Peep
authors.initials: P
authors.lastname: Palumaa
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Mario
authors.initials: M
authors.lastname: Piccioli
authors.forename: Simone
authors.initials: S
authors.lastname: Ciofi-Baffoni
authors.forename: Lucia
authors.initials: L
authors.lastname: Banci
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013455:Sulfur / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D007501:Iron