Understanding the Mechanism of [4Fe-4S] Cluster Assembly on Eukaryotic Mitochondrial and Cytosolic Aconitase

Abstract

Iron−sulfur (Fe−S) clusters are common prosthetic groups that are found within a variety of proteins responsible for functions that include electron transfer, regulation of gene expression, and substrate binding and activation. Acquisition of a [4Fe-4S] cluster is essential for the functionality of many such roles, and dysfunctions in Fe−S cluster synthesis and trafficking often result in human disease, such as multiple mitochondrial dysfunctions syndrome. While the topic of [2Fe-2S] cluster biosynthesis and trafficking has been relatively well studied, the understanding of such processes involving [4Fe-4S] centers is less developed. Herein, we focus on the mechanism of the assembly of [4Fe-4S] clusters on two members of the aconitase family, differing also in organelle placement (mitochondrion and cytosol) and biochemical function. Two mechanistic models are evaluated by a combination of kinetic and spectroscopic models, namely, a consecutive model (I), in which two [2Fe-2S] clusters are sequentially delivered to the target, and a prereaction equilibrium model (II), in which a [4Fe-4S] cluster transiently forms on a donor protein before transfer to the target. The paper also addresses the issue of cluster nuclearity for functionally active forms of ISCU, NFU, and ISCA trafficking proteins, each of which has been postulated to exist in both [2Fe-2S] and [4Fe-4S] bound states. By the application of kinetic assays and electron paramagnetic resonance spectroscopy to examine delivery pathways from a variety of potential [2Fe-2S] donor proteins to eukaryotic forms of both aconitase and iron regulatory protein, we conclude that a consecutive model following the delivery of [2Fe-2S] clusters from NFU1 is the most likely mechanism for these target proteins.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

“…Of these three, only the N6-acetyl lysine would fall outside the mitochondrial targeting sequence, and it was found using structural modeling that the nucleotide portion of SAM would fall a dis- Inasmuch as the auxiliary cluster site appeared to be almost completely occupied in the as-isolated form of the enzyme, cluster delivery must have taken place at the reducing site to yield the fully reconstituted and active form of human LIAS. Because formation of the [4Fe-4S] cluster at the reducing site is mediated by the consecutive delivery of clusters from [2Fe-2S] donors, the second step, forming the final [4Fe-4S] center, must be rate limiting and is consistent with prior observations for the [4Fe-4S] cluster protein, aconitase [24]. The highest apparent second order rate constant for cluster reconstitution was demonstrated by full-length ISCA2, followed by the truncated, but not necessarily physiological form of ISCA2, and then ISCU (Table 1).…”

“…These were chosen because they had been implicated in the trafficking of clusters to LIAS based on experimental data or observations found in patients with NKH and MMDS [19], or to the reconstitution of other [4Fe-4S]-containing proteins [21,22]. Both full-length and truncated versions of ISCA2 were utilized as [2Fe-2S] donors to further compare the reactivity between the two constructs characterized in previous reports [23,24]. A BOLA3-GLRX5 heterodimer has also been shown to have [2Fe-2S] cluster trafficking ability and has been indirectly implicated in cluster transfer to LIAS [19,25,26].…”

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

“…Hence, our in vitro results are consistent with a nonessential role for NFU proteins in plant mitochondria. Moreover, our cluster transfer studies also support mitochondrial aconitase maturation via intact [4Fe-4S] 2+ cluster transfer, rather than a mechanism involving sequential transfer of two [2Fe-2S] 2+ clusters followed by in situ reductive coupling, as recently proposed for human mitochondrial and cytosolic aconitase ( 57 ).…”

“…Moreover, Mössbauer and NMR studies have also demonstrated that reconstituted human NFU1 exclusively contains a [4Fe-4S] 2+ cluster ( 53 , 54 ). This reinterpretation necessitates a major reevaluation of the results and conclusions made by Cowan and co-workers ( 55 , 56 , 57 , 58 , 59 ) about human NFU1. The discovery that both NFU4 and NFU5 are [4Fe-4S] 2+ cluster-binding proteins, coupled with our inability to assemble a [2Fe-2S] 2+ cluster on NFU4 and NFU5 either by reconstitution or cluster transfer from [2Fe-2S]-ISCA1a/2 or [2Fe-2S]-GRXS15, is in accord with the statement that all mitochondrial NFU proteins function in [4Fe-4S] 2+ cluster trafficking.…”

“…In contrast, the human and yeast mitochondrial NFU proteins are part of the ISC machinery and have been proposed to function exclusively as [4Fe-4S] 21 cluster-carrier proteins based on both in vivo and in vitro evidence (33)(34)(35)(52)(53)(54). This hypothesis has recently been challenged in a series of five publications by Cowan and co-workers (55)(56)(57)(58)(59), which claim that human mitochondrial NFU1 is a [2Fe-2S] 21 cluster-carrier protein, based on UV-visible absorption/CD and EPR data. This interpretation is incorrect, based on our characterization of the homologous mitochondrial At NFU4 and NFU5 as [4Fe-4S] 21 cluster-carrier proteins.…”