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J. Biol. Chem., Vol. 282, Issue 42, 30586-30595, October 19, 2007

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Evidence for a Dinuclear Active Site in the Metallo--lactamase BcII with Substoichiometric Co(II)

A NEW MODEL FOR METAL UPTAKE^*

Leticia I. Llarrull¹², Mariana F. Tioni¹³⁴, Jason Kowalski, Brian Bennett⁵, and Alejandro J. Vila⁴⁶

From the Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, and Biophysics Section, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina and the National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-0509

Metallo--lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to -lactam antibiotics. Metallo--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--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--lactamase turnover.

Received for publication, June 5, 2007 , and in revised form, August 1, 2007.

^* This work was supported by grants from Agencia Nacional de Promoción de Ciencia y Tecnología of Argentina (ANPCyT) and the Howard Hughes Medical Institute (to A. J. V.). The Bruker Avance II 600-MHz NMR spectrometer was purchasedwithfundsfromANPCyT(PME2003-0026) and Consejo Nacionalde Investigaciones Científicas y Técnicas de Argentina (CONICET). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ These two authors contributed equally to this work.

² Recipient of a doctoral fellowship from CONICET.

³ Recipient of a postdoctoral fellowship and a travel grant from CONICET.

⁴ Staff members of CONICET.

⁵ Supported by National Institutes of Health Grants AI056231 and EB001980.

⁶ An International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Suipacha 531, S2002LRK Rosario, Argentina. Tel.: 54-341-435-0661 (ext. 108); Fax: 54-341-439-0465; E-mail: vila{at}ibr.gov.ar.

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