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Much remains to be elucidated concerning the selectivity mechanism of supposedly identical active sites in oligomeric proteins. Glyoxalase I (GlxI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. The E. coli GlxI is a Ni²⁺/Co²⁺-activated homodimeric protein containing two symmetric, and dually metallated active sites as characterized by X-ray structure determination. Nevertheless, kinetics and isothermal titration calorimetric (ITC) studies indicate that dimeric GlxI binds to metal ions in a ratio of 1:1 (one metal ion/one dimer) [Clugston, S. L., Yajima, R., and Honek, J. F. (2004) Biochem. J. 377, 309−316]. In the current study, we provide spectroscopic evidence for the nonequivalent metallation of GlxI by use of ¹⁵N−¹H HSQC NMR titration experiments. ¹⁵N−¹H HSQC NMR spectra reveal that the local conformations of the two active sites in homodimeric …