Formation of [4Fe-4S] Clusters in the Mitochondrial Iron–Sulfur Cluster Assembly Machinery

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.