Abstract
In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia. Recently, a mutation that suppressed the loss of the FXN homolog in Saccharomyces cerevisiae was identified that encodes an amino acid substitution equivalent to the human variant ISCU2 M140I. Here, we developed iron-sulfur cluster synthesis and transfer functional assays and determined that the human ISCU2 M140I variant can substitute for FXN in accelerating the rate of iron-sulfur cluster formation on the monothiol glutaredoxin (GRX5) acceptor protein. Incorporation of both FXN and the M140I substitution had an additive effect, suggesting an acceleration of distinct steps in iron-sulfur cluster biogenesis. In contrast to the canonical role of FXN in stimulating the formation of [2Fe-2S]-ISCU2 intermediates, we found here that the M140I substitution in ISCU2 promotes the transfer of iron-sulfur clusters to GRX5. Together, these results reveal an unexpected mechanism that replaces FXN-based stimulation of the iron-sulfur cluster biosynthetic pathway and suggest new strategies to overcome the loss of cellular FXN that may be relevant to the development of therapeutics for Friedreich's ataxia.
Highlights
In humans, mitochondrial iron–sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron–sulfur cluster assembly scaffold protein (ISCU2)
We found that human ISCU2M140I, like S. cerevisiae Isu1Sup, accelerates iron–sulfur biosynthesis in the absence of FXN in complete reconstituted reactions
The SDAecU and SDAecUM140I complexes were generated by combining recombinantly expressed human NFS1–ISD11 complex that copurified with E. coli ACP (SDAec) [6, 33] with ISCU2 or the ISCU2M140I variant
Summary
We purified native ISCU2 and the ISCU2M140I variant and probed their secondary structure, oligomeric state, and ability to form iron–sulfur assembly complexes. Similar overall rates of [2Fe–2S]–GRX5 formation were observed for the SDAecUF and SDAecUM140I complexes (Fig. 4 and Table 2), which were approximately three times greater than for the SDAecU complex The results from this in vitro iron–sulfur cluster biosynthetic assay are consistent with the stimulation of activity both by FXN and by ISCU2M140I in the absence of FXN. The SDAecUM140IF complex had a [2Fe– 2S]–GRX5 formation rate that was greater than the SDAecU complex (nearly 7-fold greater) and was approximately twice the rate of either the SDAecUF or SDAecUM140I complex (Fig. 4 and Table 2) This additive result in the iron–sulfur assembly assay hints that the stimulatory effects of FXN and ISCU2M140I occur at different steps in iron–sulfur cluster biosynthesis.
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