Arsenic binding to human metallothionein.

Area of Science:

• Biochemistry
• Toxicology
• Biophysics

Background:

• Chronic arsenic poisoning is a growing global health concern.
• Arsenic (As(3+)) readily binds to biological thiols, particularly mammalian metallothionein (MT).
• Mammalian MT is a sulfur-rich protein with two domains (alpha and beta) that bind metals.

Purpose of the Study:

• To investigate the binding stoichiometry and kinetics of As(3+) with recombinant human metallothionein (rhMT).
• To analyze the stepwise metalation reactions in both the alpha and beta domains of rhMT.
• To simulate the metalation process, providing insights into apo MT and As-MT intermediate species concentrations over time and temperature.

Main Methods:

• Utilized time- and temperature-resolved electrospray ionization mass spectrometry for speciation analysis.
• Performed kinetic analysis of As(3+) saturation reactions on isolated alpha and beta domains of rhMT.
• Quantified individual rate constants and activation energies for sequential As(3+) binding steps.

Main Results:

• As(3+) binds to rhMT with stoichiometries of As(3)S(cys9) in the beta domain and As(3)S(cys11) in the alpha domain.
• The binding reaction is noncooperative, involving three sequential bimolecular metalation steps.
• Individual rate constants and activation energies for each binding step in both domains were determined at 298 K and pH 3.5.

Conclusions:

• This study provides the first complete kinetic analysis and simulation of As(3+) binding to MT domains.
• The findings elucidate the stepwise mechanism of arsenic interaction with MT.
• Understanding these kinetics is critical for assessing the long-term effects of arsenic exposure and toxicity.