Functional transitions of the Aspergillus fumigatus iron regulator HapX are governed by conserved domains cooperatively binding [2Fe-2S] clusters.

Accurate sensing of cellular iron levels is vital, as this metal is essential but toxic in excess. The iron-sensing transcription factor HapX is crucial for virulence of Aspergillus fumigatus, the predominant human mold pathogen. Its absence impairs growth under iron limitation and excess, but not under moderate iron availability, suggesting that HapX switches between three states to adapt to varying iron availability. This study suggests that the HapX state transitions are regulated by different propensities of four phylogenetically conserved cysteine-rich regions (CRRs) to coordinate [2Fe-2S] clusters, resulting in cumulative occupancies depending on iron availability. The iron starvation state features no [2Fe-2S] clusters in any of the CRRs, the iron sufficiency/"neutral" state features clusters in CRR-B and/or -C, and the iron excess state has clusters in CRR-A, -B, and -C, while CRR-D plays a minor role. Combinatorial mutation of CRR-B and -C inhibited growth by locking HapX in the iron starvation state, leading to uncontrolled iron uptake and repression of iron-consuming pathways and iron detoxification. This growth defect was partially rescued by removing the C-terminal 27 amino acids, which are crucial for the iron starvation state and contain a degron. Noteworthy, the HapX iron starvation state induced several gene clusters encoding secondary metabolites.