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Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Cadmium is often used as a useful NMR 1 probe to study zincor calcium-containing proteins; 2 we present here a method for identification of cadmium-coordinated histidine via 1 H-15 N HMQC, which does not require any Cd excitation pulse. The cross-peaks show an E-COSY 3 type of pattern which allows the measurement of the 3 J H-Cd and 1 J N-Cd coupling constants.In many metalloproteins, notably those containing zinc or calcium, the natural metal is not conveniently detectable by NMR. To overcome this problem, substitution of the metal by cadmium has often been used. In particular, cadmium-113 NMR has often been used to study zinc-containing proteins; 2 the 113 Cd chemical shift is very sensitive to the nature, number, and geometric arrangement of the ligands within the coordination sphere. In many cases, the substitution of Zn 2+ by Cd 2+ has been shown to have only a modest effect on the catalytic activity of metalloenzymes. 4 113 Cd resonances are commonly detected by direct observation ( 113 Cd has spin 1 / 2 and a sensitivity 63% that of 13 C) or by inverse detection of 113 Cd scalar-coupled to 1 H. Cysteine, methionine, and histidine residues have been successfully identified as the coordinating ligands using 1 H-113 Cd HMQC, 113 Cdedited 1 H-1 H COSY or 1 H-113 Cd heteroTOCSY experiments. [5][6][7][8] These inverse experiments require a time-delay for transfer of magnetization between 1 H and 113 Cd spin, and also a knowledge of the 113 Cd chemical shift, in view of the very large chemical shift range of 113 Cd, which can also make direct detection difficult. These prerequisites make the experiments difficult in many cases.It would therefore be desirable to have a method by which the ligands of 113 Cd can be identified which does not rely on the polarization transfer between 1 H and 113 Cd spins. We describe here a method for identifying cadmium-coordinated histidines using a two-dimensional 1 H-15 N HMQC experiment which does not require Cd excitation pulses and which permits the identification of the histidine imidazole proton and nitrogen resonances of 113 Cd-bound imidazoles and the determination of the 3 J H-Cd and 1 J N-Cd coupling constants.We demonstrate the method using the zinc -lactamase from Bacillus cereus strain 569/H/9 (BCII). Zinc -lactamases 9 in pathogenic bacteria, which can be plasmid-encoded, confer resistance toward all the -lactam antibiotics and represent a real threat to antibiotic therapy because no inhibitors are presently available for clinical purposes. 10 The crystal structure 11 of -lactamase BCII shows two zinc cations in the active site, one (site I) coordinated by three histidines and one water molecule, and the other (site II) coordinated by a histidine, a cysteine, an aspartate, and an unknown molecule, likely to be a carbonate ion. These two zinc ions can be replaced by 113 Cd with minimal change in the kinetic parameters of the enzyme. The 113 Cd spectrum of the enzyme showed two signals, at 143 and 266 ppm relative to 0.1 M Cd-(ClO 4 ) 2 ; comparison with previously rep...