Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein.
The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.
Ngu
Thanh T
TT
Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7.
Lee
Janice A
JA
Rushton
Moira K
MK
Stillman
Martin J
MJ
D020418:Algal Proteins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001151:Arsenic / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D044645:Fucus / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D020418:Algal Proteins; D011994:Recombinant Proteins; D008668:Metallothionein; D001151:Arsenic