In an effort to further probe metal binding to metallo-beta-lactamase L1 (mbetal L1), Cu- (Cu-L1) and Ni-substituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equiv of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin-spin coupling. Cu-L1 exhibited higher k(cat) (96 s(-1)) and K(m) (224 microM) values, as compared to the values of dinuclear Zn(II)-containing L1, when nitrocefin was used as substrate. The Ni-L1 bound 1 equiv of Ni and 0.3 equiv of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by (1)H NMR spectra, which showed relatively sharp proton resonances. Stopped-flow kinetic studies showed that ZnNi-L1 stabilized significant amounts of the nitrocefin-derived intermediate and that the decay of intermediate is rate-limiting. (1)H NMR spectra demonstrate that Ni(II) binds in the Zn(2) site and that the ring-opened product coordinates Ni(II). Both Cu-L1 and ZnNi-L1 hydrolyze cephalosporins and carbapenems, but not penicillins, suggesting that the Zn(2) site modulates substrate preference in mbetal L1. These studies demonstrate that the Zn(2) site in L1 is very flexible and can accommodate a number of different transition metal ions; this flexibility could possibly offer an organism that produces L1 an evolutionary advantage when challenged with beta-lactam-containing antibiotics.