A metallothionein type 2 from Avicennia marina binds to iron and mediates hydrogen peroxide balance by activation of enzyme catalase.

Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins that are important for essential metal homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. In this work the gene encoding an MT type 2 from Avicennia marina (Forssk.) Vierh. (AmMT2) was cloned into pET41a and transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl β-_D-1-thiogalactopyranoside, AmMT2 was expressed as glutathione-S-transferase (GST)-tagged fusion protein. The accumulation of Zn²⁺, Cu²⁺, Fe²⁺, Ni²⁺ and Cd²⁺ for strain R-AmMT2 was 4, 8, 5.4, 2 and 1.6 fold of control strain suggesting the role of AmMT2 in accumulation of metals. Particularly the strain R-AmMT2 was able to accumulate 30.7 mg per g dry weight. The cells expressing AmMT2 was more tolerant to hydrogen peroxide and had higher catalase (CAT) activity. To understand the mechanistic action of AmMT2 hydrogen peroxide tolerance, the activity of CAT in the E. coli protein extract was assayed after addition of pure Fe²⁺/GST-AmMT complex and Apo/GST-AmMT2 in vitro. Whereas, the activity of CAT did not change by the addition of Apo/GST-AmMT2, the activity of CAT significantly increased after addition of Fe²⁺/GST-AmMT2. These results show that AmMT2 activates CAT through Fe²⁺ transfer which subsequently causes the oxidative stress tolerance.