Functional characterization of rice metallothionein OsMT-I-Id: Insights into metal binding and heavy metal tolerance mechanisms.

Metallothioneins (MTs) are cysteine-rich proteins known for their strong metal-binding capabilities, making them effective in detoxifying heavy metals (HMs). This study focuses on characterizing the functional properties of OsMT-I-Id, a type-I Metallothionein found in rice. Using a HM-responsive yeast cup1Δ (DTY4), ycf1∆ (for cadmium), and acr3∆ mutants (for trivalent arsenic), we assessed the impact of OsMT-I-Id on metal accumulation and cellular resilience. Our results demonstrated that yeast cells expressing OsMT-I-Id showed increased tolerance and accumulated higher levels of copper (Cu), arsenic (As), and cadmium (Cd), compared to control cells. This can be attributed to the protein's ability to chelate and bind HMs. Site-directed mutagenesis was employed to investigate the specific contributions of cysteine residues. The study revealed that yeast cells with a mutated C-domain displayed heightened HM sensitivity, while cells with a mutated N-domain exhibited reduced sensitivity. This underscores the critical role of C-cysteine-rich domains in metal binding and tolerance of type-I rice MTs. Furthermore, the study identified the significance of the 12th cysteine position at the N-domain and the 68th and 72nd cysteine positions at the C-domain in influencing OsMT-I-Id metal-binding capacity. This research provides novel insights into the structure-function relationship and metal binding properties of type-I plant MTs.