Spectroscopic Studies on Cobalt(II)-Substituted Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

Abstract

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.

Conformational Changes in the Metallo-β-lactamase ImiS During the Catalytic Reaction: An EPR Spectrokinetic Study of Co(II)-Spin Label Interactions

“…EPR and NMR spectroscopic studies have shown that the catalytically required Zn(II) (or Co(II) in substituted ImiS) is bound by a cysteine and a histidine residue and can adopt four-or fivefold coordination, likely depending on the number of solvent ligands. 19 This site corresponds to the B2 MβL consensus 'Zn2' site whose structure is known from X-ray crystallographic characterization of the very closely related B2 MβL CphA. 20 A catalytically-competent five-coordinate Co (II) reaction intermediate species was characterized by EPR spectrokinetic studies of Cosubstituted ImiS and confirmed the direct involvement of the metal ion in catalysis.…”

“…Zn-ImiS* and Co-ImiS*, containing one equivalent of Zn(II) and Co(II), respectively, were prepared and characterized as described for wild-type Zn-and Co-ImiS. 19 Steady state kinetics with imipenem as substrate were carried out at 25 °C in 50 mM Tris buffer, pH 7.0.…”

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

“…A similar binding site, on the basis of cobalt substitution, has been predicted for ImiS from Aeromonas Veronii, which shares 98% amino acid sequence identity with CphA (79). An alternative model, suggesting that the catalytic metal ion is either labile (distributed between the two sides of the active site) or resides in a site made up of ligands from both sites has been put forth for AE036 from A. hydrophila (77).…”

“…A remote binding site for the second, inhibitory equivalent of metal was previously suggested for the 2-Co analogue of ImiS, on the basis of EPR, NMR, and optical spectroscopy (79). The EXAFS data for 2-Zn ImiS are consistent with a remote binding site, on the basis of the lack of an obvious metal-metal interaction and the presence of a sulfur ligand in the coordination sphere of the inhibitory metal ion (Zn binding at the Zn 1 site would be expected to display tetracoordination, including three histidine ligands).…”

“…The B2 enzymes are less well-characterized, with comparably fewer physicochemical studies available (73)(74)(75)(76)(77)(78)(79)(80)(81). Recently, the first crystal structure of a B2 enzyme was reported (82).…”