Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of -lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo--lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark intermediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the -lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the -lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo--lactamases may be atypical and that cleavage of the -lactam amide bond is the rate-determining step for breakdown of the majority of -lactam substrates by the L1 enzyme.Zinc-dependent or metallo--lactamases (1) are bacterial enzymes of considerable clinical and mechanistic interest. In the clinical context, their ability to hydrolyze the newer generation of carbapenem antibiotics (2), together with the potential for interspecies transfer arising from their presence on mobile genetic elements (3, 4), is a cause for increasing concern as carbapenem use becomes more widespread. The group includes both nonspecific enzymes (able to efficiently hydrolyze penicillin and cephalosporin antibiotics in addition to carbapenems) and specific enzymes (5) and may utilize one or two zinc ions for maximal activity (6 -8). Although mechanistic analogies with zinc peptidases have been attempted (7), it is now clear that metallo--lactamases are members of a distinct superfamily of hydrolases that includes bacterial arylsulfatases and type II glyoxalases (8, 9). Despite increasing attention in recent years (10 -12), understanding of metallo--lactamase mechanism remains incomplete, and there are to date no clinically effective inhibitors.There is general agreement that metallo--lactamases hydrolyze substrate by a nucleophilic attack of zinc-bound water upon the carbonyl carbon of the scissile bond (Ref. 13 and references therein). This would be expected to lead to formation of a tetrahedral oxyanion intermediate that would decay to product via cleavage of the -lactam amide bond and protonation of the amide nitrogen. Several models of such a reaction scheme have been postulated (7,10,14,15); these vary primarily in the source of the proton required for product formation. Experimental evidence points to different conclusions for different enzymes: in the BCII enzyme from Bacillus cereus, a conserved aspartate in the active site appears to shuttle a proton from the oxyanion to the amide nitrogen, and the major intermedia...