Arsenic-metalation of triple-domain human metallothioneins: Support for the evolutionary advantage and interdomain metalation of multiple-metal-binding domains

Metallothionein (MT) is a prominent metal-binding protein and in mammalian systems contains a two-domain beta alpha motif, while in lower life forms MT often consists of only a single-domain structure. There are also unusual MTs from American oysters that consist of multiple domains (from one to four alpha domains). This report details the study of the As3+-metalation to two different concatenated triple beta and alpha domain MTs using time-resolved electrospray ionization mass spectrometry (ESI MS). Analysis of kinetic ESI MS data show that alpha alpha alpha human MT and beta beta beta human MT bind As3+ in a noncooperative manner and involves up to 11 sequential bimolecular reactions. We report the complete progress of the reactions for the As3+-metalation of both triple-domain MTs from zero and up to 9 (beta beta beta) or 10 As3+ ions (alpha alpha alpha). The rate constants for the As3+-metalation are reported for both the beta beta beta and alpha alpha alpha human MT. At room temperature (298 K) and pH 3.5, the sequential individual rate constants, k(n) (n = 1-9) for the As3+-metalation of beta beta beta hMT starting at k(1 beta beta beta) are 40, 36, 37, 26, 27, 17, 12, 6, and 1 M-1 s(-1); while at room temperature (298 K) and pH 3.5, the sequential individual rate constants, k(n) (n = 1-10) for the As3+-metalation of alpha alpha alpha hMT starting at k(1 alpha alpha alpha) are 52, 45, 46, 42, 38, 36, 29, 25, 14, and 6 M-1 s(-1). The trend in the rate constant values reported for these two triple-domain MT proteins supports our previous proposal that the rate constant values are proportionally related to the total number of equivalent binding sites. The rate of binding for the 1st As3+ is the fastest we have determined for any MT to date. Additionally, we propose that the data show for the first time for any MT species, that interdomain metalation occurs in the binding of the 10th and 11th As3+ to alpha alpha alpha hMT. (C) 2010 Elsevier Inc. All rights reserved.