Proton Transfer Pathways between Active Sites and Proximal Clusters in the Membrane-Bound [NiFe] Hydrogenase

[NiFe] hydrogenases are enzymes that catalyze the splitting of molecular hydrogen according to the reaction H₂ → 2H⁺ + 2e^-. Most of these enzymes are inhibited even by low traces of O₂. However, a special group of O₂-tolerant hydrogenases exists. A member of this group is the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha ( ReMBH). The ReMBH harbors an unusual iron sulfur cluster with composition 4Fe3S(6Cys) that is able to undergo structural changes triggering the flow of two electrons to the [NiFe] active site. These electrons promote oxygen reduction at the active site, preventing, in this way, aerobic inactivation of the enzyme. In the superoxidized state, the [4Fe3S] cluster binds to a hydroxyl group that originates from either molecular oxygen or water reaching the site. Both reactions, oxygen reduction to water at the [NiFe]- or [4Fe3S]-centers and oxygen evolution from water at the proximal cluster, require the delivery of protons regulated by a subtle communication mechanism between these metal centers. In this work, we sequentially apply multiscale modeling techniques as quantum mechanical/molecular mechanics methods and classical molecular dynamics simulations to investigate the role of two distinct proton transfer pathways connecting the [NiFe] active site and the [4Fe3S] proximal cluster of ReMBH in the protection mechanism against an oxygen attack. Although the "glutamate" pathway is preferred by protons migrating toward the active site to avoid inactivation by O₂, the "histidine" pathway plays an essential role in delivering protons for O₂ reduction at the proximal cluster. The results obtained in this work not only provide new pieces to the puzzling catalytic mechanisms governing O₂-tolerant hydrogenases but also highlight the relevance of dynamics in the proper description of biochemical reactions in general.