A [FeFe] Hydrogenase–RubrerythrinChimericEnzyme Functions to Couple H₂ Oxidation to Reduction ofH₂O₂ in the Foodborne Pathogen Clostridium perfringens

[FeFe] hydrogenases are a diverse class of H₂-activating enzymes with a wide range of utilities in nature. As H₂ 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 H₂O₂ detoxification, raises the question of whether catalytic reactions, such as H₂ oxidation and H₂O₂ reduction, are functionally linked. Herein, we express and purify a representative chimera from Clostridium perfringens (termed <i>Cper</i>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)₄ centers, and a hemerythrin-like diiron site. The absence of an H₂-evolution current in protein film voltammetry highlights an exceptional bias of this enzyme toward H₂ oxidation to the greatest extent that has been observed for a [FeFe] hydrogenase. Here, we demonstrate that <i>Cper</i>HydR uses H₂, catalytically split by the hydrogenase domain, to reduce H₂O₂ by the diiron site. Structural modeling suggests a homodimeric nature of the protein. Overall, this study demonstrates that <i>Cper</i>HydR is an H₂-dependent H₂O₂ reductase. Equipped with this information, we discuss the possible role of this enzyme as a part of the oxygen-stress response system, proposing that <i>Cper</i>HydR constitutes a new pathway for H₂O₂ mitigation.