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

Abstract

Metallothionein (MT) is a prominent metal-binding protein and in mammalian systems contains a two-domain betaalpha 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 As(3+)-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 alphaalphaalpha human MT and betabetabeta human MT bind As(3+) in a noncooperative manner and involves up to 11 sequential bimolecular reactions. We report the complete progress of the reactions for the As(3+)-metalation of both triple-domain MTs from zero and up to 9 (betabetabeta) or 10As(3+) ions (alphaalphaalpha). The rate constants for the As(3+)-metalation are reported for both the betabetabeta and alphaalphaalpha human MT. At room temperature (298K) and pH3.5, the sequential individual rate constants, k(n) (n=1-9) for the As(3+)-metalation of betabetabetahMT starting at k(1betabetabeta) are 40, 36, 37, 26, 27, 17, 12, 6, and 1M(-1)s(-1); while at room temperature (298K) and pH3.5, the sequential individual rate constants, k(n) (n=1-10) for the As(3+)-metalation of alphaalphaalphahMT starting at k(1alphaalphaalpha) are 52, 45, 46, 42, 38, 36, 29, 25, 14, and 6M(-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 As(3+) 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 As(3+) to alphaalphaalphahMT.