Title

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

Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

4-2005

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

doi: 10.1021/bi047463s

Abstract

In an effort to probe the structure of a group Bb metallo-β-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. 1H 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 Zn2 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 Zn1 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-β-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.

Comments

Biochemistry, Vol. 44, No. 13 (April 2005): 5168-5176. DOI.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.