Mapping cellular Fe–S cluster uptake and exchange reactions – divergent pathways for iron–sulfur cluster delivery to human ferredoxins

Abstract

Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe–2S] or [4Fe–4S] clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe–2S] cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular cluster trafficking. This is the first step toward building a network map for all of the possible iron-sulfur cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

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.…”

“…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). Minor peak shifts were seen, but the overall signal mainly resembled Isa1, indicating possible formation of a heterodimeric complex.…”

“…As found for native human NFU1, cluster transfer to ferredoxins 1 and 2 (Fdx1 and Fdx2) [1] and glutaredoxins 2 and 3 (Grx2 and Grx3) was observed [10]. Cluster transfer was monitored by use of CD spectroscopy because unique spectra are generated for each iron-sulfur cluster bound protein [48].…”