Protonation state and substrate binding to B2 metallo-beta-lactamase CphA from Aeromonas hydrofila.

The zinc enzymes metallo beta-lactamases counteract the beneficial action of beta-lactam antibiotics against bacterial infections, by hydrolyzing their beta-lactam rings. To understand structure/function relationships on a representative member of this class, the B2 M beta L CphA, we have investigated the H-bond pattern at the Zn enzymatic active site and substrate binding mode by molecular simulation methods. Extensive QM calculations at the DFT-BLYP level on eleven models of the protein active site, along with MD simulations of the protein in aqueous solution, allow us to propose two plausible protonation states for the unbound enzyme, which are probably in equilibrium. Docking procedures along with MD simulations and QM calculations suggest that in the complex between the enzyme and its substrate (biapenem), the latter is stable in only one of the two protonation states, in addition it exhibits two different binding modes, of which only one agrees with previous proposals. In both cases, the substrate is polarized as in aqueous solution. We conclude that addressing mechanistic issues on this class of enzymes requires a careful procedure to assign protonation states and substrate docking modes.

Cis-trans isomerization in triply-bonded ditungsten complexes: a multitude of possible pathways.

We have investigated different possible mechanisms for the cis-trans isomerization in triply bonded ditungsten complexes with stoichiometry W(2)Cl(4)(NHEt)(2)(PMe(3))(2) using static density functional calculations as well as Car-Parrinello simulations. Our studies reveal an unexpected richness of possible reaction pathways that include both unimolecular and bimolecular mechanisms. Among the possible routes that have been identified are processes involving successive dissociation/reassociation of phosphine ligands, intramolecular chloride hopping, intertungsten phosphine exchange as well as numerous combinations of these basic reaction types. All pathways involve maximal activation barriers of less than 35 kcal/mol and include phosphine concentration dependent and independent routes. The energetically most favorable phosphine-dependent pathway is based on the dissociation/reassociation of phosphine ligands. This path is characterized by a maximal dissociation barrier of 18 kcal/mol. The fastest alternative unimolecular route (with a maximal activation barrier of 24 kcal/mol) is based on a direct exchange of phosphine between the two metallic coordination centers. All the identified pathways, with the exception of a previously proposed internal flip mechanism that can be ruled out on energetic grounds, are competitive and may contribute in various combinations to the overall reaction rate. The identified isomerization mechanisms are fully consistent with the experimentally observed 3-state-kinetics and the dependence of the overall reaction rate on the excess concentration of phosphine which is demonstrated with a simplified kinetic model of the process.

Three- and four-center trans effects in triply bonded ditungsten complexes: an ab initio molecular dynamics study of compounds with stoichiometry W2Cl4(NHEt)2(PMe3)2.

We have performed ab initio molecular dynamics simulations based on density functional theory to characterize the structural, electronic, and dynamic properties of the three major isomeric forms of the title compound. In agreement with experimental results, calculations with two different parametrizations of the exchange-correlation functional (BLYP and BP) both indicate the cis-C2 form as the most stable isomer. The relative energies of the different forms are, however, small (less than approximately 1-2 kcal/mol), and the three compounds show overall very similar ground-state properties. Larger differences exist in their finite temperature behavior, which is dominated by the facile dissociation of one or both phosphine ligands. The calculated activation energies for phosphine dissociation differ clearly for the trans and the cis isomers and vary in the order trans << cis-C2 less than approximately cis-Ci. Analysis of the electronic structure of the transition states shows that the difference in activation energy between cis and trans isomers can be rationalized in terms of a classic trans effect caused by a molecular orbital spanning the three atomic centers N-W-P. The subtle difference between the two cis isomers, on the other hand, is likely due to an analogous four-center trans effect N-W-W-P which is mediated via metal-metal orbitals and involves ligands on both tungsten atoms.