Inactivation Mechanism of Glycerol Dehydration by Diol Dehydratase from Combined Quantum Mechanical/Molecular Mechanical Calculations

Inactivation of diol dehydratase during the glycerol dehydration reaction is studied on the basis of quantum mechanical/molecular mechanical calculations. Glycerol is not a chiral compound but contains a prochiral carbon atom. Once it is bound to the active site, the enzyme adopts two binding conformations. One is predominantly responsible for the product-forming reaction (GR conformation), and the other primarily contributes to inactivation (GS conformation). Reactant radical is converted into a product and byproduct in the product-forming reaction and inactivation, respectively. The OH group migrates from C2 to C1 in the product-forming reaction, whereas the transfer of a hydrogen from the 3-OH group of glycerol to C1 takes place during the inactivation. The activation barrier of the hydrogen transfer does not depend on the substrate-binding conformation. On the other hand, the activation barrier of OH group migration is sensitive to conformation and is 4.5 kcal/mol lower in the GR conformation than in the GS conformation. In the OH group migration, Glu170 plays a critical role in stabilizing the reactant radical in the GS conformation. Moreover, the hydrogen bonding interaction between Ser301 and the 3-OH group of glycerol lowers the activation barrier in GR-TS2. As a result, the difference in energy between the hydrogen transfer and the OH group migration is reduced in the GS conformation, which shows that the inactivation is favored in the GS conformation

Catalytic Roles of the Metal Ion in the Substrate-Binding Site of Coenzyme B₁₂-Dependent Diol Dehydratase

Functions of the metal ion in the substrate-binding site of diol dehydratase are studied on the basis of quantum mechanical/molecular mechanical (QM/MM) calculations. The metal ion directly coordinates to substrate and is essential for structural retention and substrate binding. The metal ion has been originally assigned to the K+ ion; however, QM/MM computations indicate that Ca2+ ion is more reasonable as the metal ion because calculated Ca−O distances better fit to the coordination distances in X-ray crystal structures rather than calculated K−O distances. The activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion. For example, the spectator OH group of substrate is fully deprotonated by Glu170 in the transition state for the OH group migration in the Ca-contained QM/MM model, and therefore the barrier height is significantly decreased in the model having Ca2+ ion. On the other hand, the deprotonation of the spectator OH group cannot effectively be triggered by the K+ ion. Moreover, in the hydrogen recombination, the most energy-demanding step is more favorable in the Ca-contained model. The proposal that the Ca2+ ion should be involved in the substrate-binding site is consistent with an observed large deuterium kinetic isotope effect of 10, which indicates that C−H bond activation is involved in the rate-determining step. Asp335 is found to have a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O−C bond cleavage by Ca2+ ion and the deprotonation of the spectator OH group by Glu170 is required to overcome the anticatalytic effect of Asp335

Possible Peroxo State of the Dicopper Site of Particulate Methane Monooxygenase from Combined Quantum Mechanics and Molecular Mechanics Calculations

Enzymatic methane hydroxylation is proposed to efficiently occur at the dinuclear copper site of particulate methane monooxygenase (pMMO), which is an integral membrane metalloenzyme in methanotrophic bacteria. The resting state and a possible peroxo state of the dicopper active site of pMMO are discussed by using combined quantum mechanics and molecular mechanics calculations on the basis of reported X-ray crystal structures of the resting state of pMMO by Rosenzweig and co-workers. The dicopper site has a unique structure, in which one copper is coordinated by two histidine imidazoles and another is chelated by a histidine imidazole and primary amine of an N-terminal histidine. The resting state of the dicopper site is assignable to the mixed-valent Cu^ICu^II state from a computed Cu–Cu distance of 2.62 Å from calculations at the B3LYP-D/TZVP level of theory. A μ-η²:η²-peroxo-Cu^II₂ structure similar to those of hemocyanin and tyrosinase is reasonably obtained by using the resting state structure and dioxygen. Computed Cu–Cu and O–O distances are 3.63 and 1.46 Å, respectively, in the open-shell singlet state. Structural features of the dicopper peroxo species of pMMO are compared with those of hemocyanin and tyrosinase and synthetic dicopper model compounds. Optical features of the μ-η²:η²-peroxo-Cu^II₂ state are calculated and analyzed with TD-DFT calculations