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.