Evidence for a Dinuclear Active Site in the Metallo-β-lactamase BcII with Substoichiometric Co(II): A New Model for Uptake

Authors

Leticia l. Llarrull, University of Notre Dame
Mariana F. Tioni, Universidad Nacional de Rosario
Jason M. Kowalski, Medical College of WisconsinFollow
Brian Bennett, Marquette UniversityFollow
Alejandro J. Vila, Universidad Nacional de Rosario

Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

10-19-2007

Publisher

American Society for Biochemistry and Molecular Biology

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Original Item ID

doi: 10.1074/jbc.M704613200

Abstract

Metallo-β-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to β-lactam antibiotics. Metallo-β-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-β-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His¹¹⁶, His¹¹⁸, and His¹⁹⁶) and DCH (Asp¹²⁰, Cys²²¹, and His²⁶³) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-β-lactamase turnover.

Comments

Published version. Journal of Biological Chemistry, Vol. 282, No. 42 (October 19, 2007): 30586-30595. DOI. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Used with permission.

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

Recommended Citation

Llarrull, Leticia l.; Tioni, Mariana F.; Kowalski, Jason M.; Bennett, Brian; and Vila, Alejandro J., "Evidence for a Dinuclear Active Site in the Metallo-β-lactamase BcII with Substoichiometric Co(II): A New Model for Uptake" (2007). Physics Faculty Research and Publications. 89.
https://epublications.marquette.edu/physics_fac/89