Aspergillus fumigatus Metallothionein CmtA Binds and Receives a [2Fe-2S] Cluster from Monothiol Glutaredoxin GrxD

Iron homeostasis is essential for the virulence of the opportunistic fungal pathogen Aspergillus fumigatus. The cytosolic monothiol glutaredoxin GrxD was recently shown to play a critical role in A. fumigatus iron metabolism via regulation of iron–sulfur (Fe–S) binding iron-responsive transcription factors and interaction with components of the cytosolic Fe–S cluster assembly pathway. Interestingly, the putative copper-binding metallothionein CmtA was also identified as a binding partner for GrxD; however, the metal-binding properties of both proteins and the nature of their interactions were unclear. Here, we addressed these open questions by recombinant expression and purification of GrxD and CmtA and analysis of their metal–protein and protein–protein interactions. Using elemental analysis and UV–visible absorption, circular dichroism, EXAFS, Mössbauer and EPR spectroscopies, we demonstrate that recombinant CmtA expressed in Escherichia coli can bind a [2Fe-2S]2+ cluster, mononuclear Fe2+, Zn2+, and Cu+, while recombinant GrxD ligates a mixture of [2Fe-2S]2+ and [3Fe-4S]+ clusters at its dimer interface. Furthermore, we discovered that the interaction between GrxD and CmtA involves thermodynamically-driven transfer of a [2Fe-2S]2+ cluster from GrxD to CmtA. Accordingly, coexpression of GrxD with CmtA in E. coli increases in vivo Fe–S cluster loading in CmtA, supporting a role for GrxD in delivery of [2Fe-2S]2+ clusters to CmtA. Taken together, these results suggest that the sole metallothionein in A. fumigatus represents a unique class of fungal metallothioneins that functionally interacts with Fe–S cluster trafficking and storage proteins.