A theoretical study of methanol oxidation mechanisms by methanol dehydrogenase enzymes for fuel cell applications

Enzymes are considered for electrochemical generation of power in fuel cells. Methanol dehydrogenase (MDH) is one such enzyme, which has been used as an anodic catalyst for a methanol-fed biofuel cell producing enough power for small electronic device applications. In practice, however, there are power output limitations associated with this MDH fuel cell, which may potentially be eliminated or reduced if the reactivity of this enzyme during the oxidation of methanol at the molecular level is clearly understood. Two mechanisms for the methanol oxidation process by MDH have been proposed in the literature, Addition-Elimination (A-E) and Hydride Transfer (H-T), but no agreement has been reached about what mechanism actually operates in reality. Also, it was suggested that ion-modified MDH, particularly Ba 2+-MDH enzyme, is more active towards oxidation of methanol than Ca 2+-MDH from experimental kinetic observations. In this dissertation, MDH active site models of varying sizes were tested for the A-E and H-T methanol oxidation in the presence of both Ca2+ and Ba2+. Potential energy surfaces for the reactions were calculated, and the feasibility of the suggested reaction mechanisms was judged by comparison with available experimental free energy barriers. By systematically increasing the size of the models, deeper insight into the details of the reactions was obtained, and the role of the various active site residues was also analyzed. Comparison of free energy barriers calculated for the rate-determining steps in this work for the A-E and H-T oxidation mechanisms with experimental Gibbs energy of activation by Ca2+-MDH showed that these two mechanisms may not be correctly proposed in the literature. Also, the reduction of barriers for the rate-determining steps in the presence of Ba2+ for A-E and H-T obtained with the best MDH active site model tested here is almost twice as much the experimental free energy reduction with Ba 2+-MDH for methanol oxidation. A modified first step of original H-T resulted in a newly proposed two-step H-T oxidation mechanism, where the barrier for the formation of final product, formaldehyde, during the first step is very much comparable (11.4 kcal/mol) to the experimental Gibbs energy of activation (8.5 kcal/mol). In the case of Ba2+ presence during this new mechanism, the free energy barrier is 6.2 kcal/mol, which is comparable to free energy of activation for oxidation of methanol by Ba2+-MDH (3.5 kcal/mol). The second and final step involving proton transfer in this Two-step H-T was observed to be mediated by a water molecule in the presence of both ions. Moreover, DFT-MD investigations on the reactant complex and necessary intermediates associated with all mechanisms also lead us to the conclusion that oxidation of methanol by MDH has a greater probability of proceeding through the two-step hydride transfer mechanism compared to proposed A-E and H-T