A [FeFe] Hydrogenase-Rubrerythrin Chimeric Enzyme Functions to Couple H Oxidation to Reduction of HO in the Foodborne Pathogen .

[FeFe] hydrogenases are a diverse class of H-activating enzymes with a wide range of utilities in nature. As H is a promising renewable energy carrier, exploration of the increasingly realized functional diversity of [FeFe] hydrogenases is instrumental for understanding how these remarkable enzymes can benefit society and inspire new technologies. In this work, we uncover the properties of a highly unusual natural chimera composed of a [FeFe] hydrogenase and rubrerythrin as a single polypeptide. The unique combination of [FeFe] hydrogenase with rubrerythrin, an enzyme that functions in HO detoxification, raises the question of whether catalytic reactions, such as H oxidation and HO reduction, are functionally linked. Herein, we express and purify a representative chimera from (termed HydR) and apply various electrochemical and spectroscopic approaches to determine its activity and confirm the presence of each of the proposed metallocofactors. The cumulative data demonstrate that the enzyme contains a surprising array of metallocofactors: the catalytic site of [FeFe] hydrogenase termed the H-cluster, two [4Fe-4S] clusters, two rubredoxin Fe(Cys) centers, and a hemerythrin-like diiron site. The absence of an H-evolution current in protein film voltammetry highlights an exceptional bias of this enzyme toward H oxidation to the greatest extent that has been observed for a [FeFe] hydrogenase. Here, we demonstrate that HydR uses H, catalytically split by the hydrogenase domain, to reduce HO by the diiron site. Structural modeling suggests a homodimeric nature of the protein. Overall, this study demonstrates that HydR is an H-dependent HO reductase. Equipped with this information, we discuss the possible role of this enzyme as a part of the oxygen-stress response system, proposing that HydR constitutes a new pathway for HO mitigation.