Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis.

Metal ions in metallo-β-lactamases (MBLs) play a major role in catalysis. In this study we investigated the role of the metal ions in the Zn1 and Zn2 sites of MBL L1 during catalysis. A ZnCo (with Zn(II) in the invariant Zn1 site and Co(II) in the Zn2 site) analog of MBL L1 was prepared by using a biological incorporation method. Extended X-ray Absorption Fine Structure (EXAFS) spectroscopic studies were used to confirm that the ZnCo analog was prepared. To study the roles of the Zn(II) and Co(II) ions during catalysis, rapid freeze quench (RFQ)-EXAFS studies were used to probe the reaction of the ZnCo-L1 analog with chromacef when quenched at 10 ms, 50 ms, and 100 ms. The L1-product complex was also analyzed with EXAFS spectroscopy. The data show that the Zn-Co distance is 3.49 Å in the resting enzyme and that this distance increases by 0.3 Å in the sample that was quenched at 10 ms. The average Zn-Co distance decreases at the other time points until reaching a distance of 3.58 Å in the L1-product complex. The data also show that a Co-S interaction is present in the 100 ms quenched sample and in the L1-product complex, which suggests that there is a significant rearrangement of product in the active site.