Characterization of the bottlenecks and pathways for inhibitor dissociation from [NiFe] hydrogenase

[NiFe] hydrogenases can act as efficient catalysts for hydrogen oxidation and biofuel production. However, some [NiFe] hydrogenases are inhibited by gas molecules present in the environment, such as O2 and CO. One strategy to engineer [NiFe] hydrogenases to achieve O2 and CO-tolerant enzymes is by introducing point mutations to block the access of inhibitors to the catalytic site. In this work, we characterized the unbinding pathways of CO in complex with the wild type and 10 different mutants of [NiFe] hydrogenase from Desulfovibrio fructosovorans using τ-Random Accelerated Molecular Dynamics (τRAMD) to enhance the sampling of unbinding events. The residence times computed with τRAMD are in agreement with the experimental ones. Extensive data analysis of the simulations revealed that, from the two bottlenecks proposed in previous studies for the transit of gas molecules (residues 74 and 122, and residues 74 and 476), only one of them (residues 74 and 122) effectively modulates diffusion and residence times for CO. We also computed pathway probabilities for the unbinding of different gas molecules from the wild type [NiFe] hydrogenase and we observed that, while the most probable pathways are the same, the secondary pathways are different. We propose that mutations to block the most probable paths, in combination with mutations to open the main secondary path used by H2, can be a feasible strategy to achieve CO and O2 resistance in the [NiFe] hydrogenase from Desulfovibrio fructosovorans.