Cytosolic iron–sulfur cluster transfer—a proposed kinetic pathway for reconstitution of glutaredoxin 3

Abstract

Iron-sulfur clusters are ubiquitously conserved and play essential cellular roles. The mechanism of Fe-S cluster biogenesis involves multiple proteins in a complex pathway. Cluster biosynthesis primarily occurs in the mitochondria, but key Fe-S proteins also exist in the cytosol. One such protein, glutaredoxin 3 (Grx3), is involved in iron regulation, sensing and mediating [2Fe-2S] cluster delivery to cytosolic protein targets, but the cluster donor for cytosolic Grx3 has not been elucidated. Herein, we delineate the kinetic transfer of [2Fe-2S] clusters into Grx3 from potential cytosolic carrier/scaffold proteins, IscU and Nfu, to evaluate a possible model for Grx3 reconstitution in vivo.

The NMR contribution to protein–protein networking in Fe–S protein maturation

“…Which is the system donating [2Fe–2S] 2+ clusters to the apo complex is still under investigation. A possible pathway, that has been recently proposed, involves the mitochondrial ISC assembly machinery: the de novo biosynthesis of the [2Fe–2S] 2+ cluster on mitochondrial ISCU2 is followed by cluster export, as [2Fe–2S](GS) 4 complex, via a membrane transporter; then, the [2Fe–2S] cluster is believed to be uptaken by the cytosolic form of ISCU2, which delivers it to cytosolic form of NFU1, which finally transfers the cluster to GLRX3 [189–192]. This molecular process is, however, only based on in vitro information, and experimental evidences from in vivo data are required to definitively validate it.…”

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)—Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway

“…Also, in contrast to native protein, the G208C variant is unable to take up the [2Fe-2S](GS) 4 complex [1]. Likewise, the mutant protein is unable to receive a [2Fe-2S] cluster from typical iron-sulfur scaffold proteins such as Isa1 and IscU (Table 5), which have been shown to deliver a cluster into native NFU1 [10, 14]. Current models for Fe/S cluster biogenesis indicate that transfer from IscU is promoted with assistance from heat shock chaperone proteins [16, 37, 47, 55].…”

“…The point mutation results in a changed preference for the glutaredoxins. Our previous work has indicated that [2Fe-2S] cluster from native NFU1 to apo Grx3 was a kinetic sink at 36000 M −1 min −1 , while transfer to Grx2 occurred, but at around the same level as other transfers from NFU1 (Table 5) [10, 14]. However, the mutant form of the protein now kinetically prefers cluster transfer to Grx2 by increasing the second-order rate constant seven-fold over transfer to Grx3, which decreased by three-fold.…”

“…We have previously demonstrated that [2Fe-2S] cluster proteins are able to transfer an iron-sulfur cluster to native human NFU1 [10, 14]. Therefore, we have examined the ability of the G208C mutant protein to be reconstituted in the same way using CD spectroscopy.…”

“…With the chaperones, IscU was unable to transfer cluster in to either the native or the mutant (Fig. S4), even though transfer from IscU to the native protein can proceed without the chaperone system [14]. Similarly, we monitored the ability of S. pombe Isa1 to transfer a cluster into G208C NFU1, since that transfer was also observed for native NFU1 [10]; however, the appearance of the G208C NFU1 signature peaks was not observed, even over the course of 3 h (data not shown).…”