Bi(III) Binding Stoichiometry and Domain-Specificity Differences Between Apo and Zn(II)-bound Human Metallothionein...

Area of Science:

• Biochemistry
• Metalloprotein Chemistry
• Toxicology

Background:

• Bismuth (Bi(III)) is a xenobiotic metal with therapeutic uses.
• Metallothioneins (MTs) are cysteine-rich proteins binding metals like Zn(II), Cd(II), and Cu(I).
• Bi(III) binding to MTs is complex, with conflicting reports on stoichiometry and pathway, influenced by pH and initial metal loading.

Purpose of the Study:

• To determine binding constants of [Bi(EDTA)]- to apo-MT1a and its αMT fragment using ESI-MS.
• To investigate domain specificity of Bi(III) binding to MT.
• To elucidate the effect of initial metal-loading status on Bi(III)-MT stoichiometry.

Main Methods:

• Electrospray ionization mass spectrometry (ESI-MS) for binding constants.
• Proteolysis methods for domain specificity.
• Titration of [Bi(EDTA)]- into apo-MT and Zn7MT.

Main Results:

• [Bi(EDTA)]- binding constants were determined for apo-MT1a and αMT fragment.
• Bi(III) binding involves cysteines spanning across traditional metal-binding domains.
• Stoichiometry of Bi(III)-MT formation differs based on initial MT structure (Bi6MT for apo-MT, Bi7MT for Zn7MT).

Conclusions:

• The initial structure of metallothionein dictates the stoichiometry of bismuth binding.
• This finding resolves discrepancies in previous Bi(III)-MT binding studies.
• Understanding Bi(III)-MT interactions is crucial for its therapeutic applications and toxicological assessment.