A [FeFe] Hydrogenase–Rubrerythrin Chimeric Enzyme Functions to Couple H2 Oxidation to Reduction of H2O2 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 CperHydR) 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 CperHydR 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 CperHydR 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 CperHydR constitutes a new pathway for H2O2 mitigation.