Uncovering the copper(I) binding abilities of a unique fungal metallothionein: Characterization of Yarrowia lipolytica MT and its Y54C mutant

Abstract

Metallothioneins (MTs) are a superfamily of cysteine-rich proteins essential for metal homeostasis and detoxification. While mammalian and plant MTs have been extensively characterized, fungal MTs remain comparatively understudied despite their remarkable sequence diversity. In particular, members of the fungal-IV MT family from Yarrowia lipolytica exhibit unusual sequence features, including a conserved CCC motif and an extended cysteine-free N-terminal region, raising the question of whether all cysteine residues participate equally in Cu(I) coordination and how sequence context influences metal binding. To address this, we investigated one representative protein (Ylip_MT) using a combination of potentiometric titrations and complementary spectroscopic techniques to resolve the stepwise recruitment of cysteine ligands during Cu(I) coordination. Truncated variants were employed to probe the role of the cysteine-free N-terminal segment, while a Y54C mutant was introduced independently to restore the otherwise conserved C-terminal cysteine and assess its influence on metal binding. Our results indicate the formation of a Cu₄Cys_8-9 cluster and demonstrate that both the extended cysteine-free N-terminal region and the C-terminal environment modulate cluster stability. This work represents the first application of potentiometry to a Cu(I)-MT system and provides new insight into how sequence-specific features govern metal binding in fungal MTs.