Metal preference of Zn(ii) and Co(ii) for the dinuclear metal binding site of IMP-1 metallo-β-lactamase and spectroscopic properties of Co(ii)-substituted IMP-1 with mercaptoacetic acid

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

“…Further addition of Co­(II) to this sample resulted in a monotonic increase in absorbance in both regions, maximizing at 2.0 equiv of added Co­(II), consistent with binding distributed between the Zn 1 and Zn 2 sites, even at very low concentrations . The final spectra (ε 330 = 1060, ε 550 = 505, ε 615 = 285 M –1 cm –1 for two Co­(II)/protein) are indistinguishable from those reported for many other di-Co­(II) B1 MβLs. ,,,,,− …”

“… 22 The final spectra (ε 330 = 1060, ε 550 = 505, ε 615 = 285 M –1 cm –1 for two Co(II)/protein) are indistinguishable from those reported for many other di-Co(II) B1 MβLs. 18 , 22 , 24 , 32 , 35 , 37 − 40 …”

“…However, in this case, maximum absorbance of the LMCT (ε = 900 M −1 cm −1 ) is achieved with 1 equiv of added Co(II), providing strong evidence that it is bound primarily at the consensus Zn 2 site, forming ZnCo-NDM-1. 35 In contrast, when the apoenzyme is incubated with 1 equiv of Cd(II) followed by titration with Co(II), the LMCT at 330 nm is nearly absent. The optical spectrum of CoCd-NDM-1 (Figure 2A, bottom) shows only a nearly symmetric pattern of four principal ligand field bands, Closer inspection of the ligand-field regions for ZnCo-NDM-1 and CoCd-NDM-1 (Figure 2B, top) further supports the description of these samples as homogeneous heterobimetallic enzymes.…”

“…Previously reported optical spectra for Co­(II)-substituted NDM-1 were obtained from samples generated by addition of Co­(II) to ZnZn-NDM-1 containing 1 mM EDTA. , With the new method to generate metal-substituted forms of NDM-1 presented here, we obtained quantifiable optical spectra of ZnCo-, CoCo-, and CoCd-NDM-1 by titration. Addition of Co­(II) to apo-NDM-1 suggests that cobalt distributes between the Zn 1 and Zn 2 sites at substoichiometric concentrations (Figure A, top), as it does in the B1MβLs BcII, , Bla2, and IMP-1 and as has been shown previously for NDM-1 . The ligand field bands between 500 and 650 nm are apparent with as little as 0.25 equiv of added Co­(II).…”

“…Added Co­(II) leads directly to the appearance of both the LMCT band at 330 nm and a broad, asymmetric set of ligand field bands (ε 550 = 220 and ε 615 = 115 M –1 cm –1 ). However, in this case, maximum absorbance of the LMCT (ε = 900 M –1 cm –1 ) is achieved with 1 equiv of added Co­(II), providing strong evidence that it is bound primarily at the consensus Zn 2 site, forming ZnCo-NDM-1 . In contrast, when the apoenzyme is incubated with 1 equiv of Cd­(II) followed by titration with Co­(II), the LMCT at 330 nm is nearly absent.…”