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Vertebrate glutaredoxin 3 (PICOT) is essential for the maturation of the heme cofactor of hemoglobin through its essential functions in iron homeostasis. The data suggest an evolutionarily conserved role of cytosolic monothiol multidomain Grxs in cellular iron metabolism pathways.

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Monothiol glutaredoxins (Grxs) with a noncanonical CGFS active site are found in all kingdoms of life. They include members with a single domain and thioredoxin-Grx fusion proteins. In Saccharomyces cerevisiae, the multidomain Grx3 and Grx4 play an essential role in intracellular iron trafficking. This crucial task is mediated by an essential Fe/S cofactor. This study shows that this unique physiological role cannot be executed by single domain Grxs, because the thioredoxin domain is indispensable for function in vivo. Mutational analysis revealed that a CPxS active site motif is fully compatible with Fe/S cluster binding on Grx4, while a dithiol active site results in cofactor destabilization and a moderate impairment of in vivo function. These requirements for Fe/S cofactor stabilization on Grx4 are virtually the opposite of those previously reported for single domain Grxs. Grx4 functions as iron sensor for the iron-sensing transcription factor Aft1 in S. cerevisiae. We found that Aft1 binds to a conserved binding site at the C-terminus of Grx4. This interaction is essential for the regulation of Aft1. Collectively, our analysis demonstrates that the multidomain monothiol Grxs form a unique protein family distinct from that of the single domain Grxs.