Arsenic Metalation of Seaweed Fucus vesiculosus Metallothionein: The Importance of the Interdomain Linker in Metallothionein†s

摘要

The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protectingnagent against toxic metals in the contaminated waters it can grow in.We report the first kinetic pathway datanfor Asn3þ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time andntemperature dependence of the relative concentrations of apo-rfMT and the five As-containing species havenbeen determined following mixing of Asn3þ and apo-rfMT using electrospray ionization mass spectrometryn(ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for Asn3þ metalation allows thensimulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consump-ntion of As1-toAs4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed herenprovides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and thenCys9 domain. The rate constants (M-1nsn-1n) calculated from the fits for the 7-cysteine γ domain are k1γ, 19.8,nand k2γ, 1.4, and for the 9-cysteine β domain are k1β, 16.3, k2β, 9.1, and k3β, 2.2. The activation energies andnArrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which asnwe show fromanalysis of the ESIMS data, allows each of its two domains to bind Asn3þ independently of eachnother. The analysis provides for the first time an explanation of the differing metal-binding properties of two-ndomainMTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers)nand mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker