Oxygen Tolerance in the [FeFe] Hydrogenase Cb HydA1

Abstract

Hydrogenases are enzymes that catalyze the formation and oxidation of hydrogen. [FeFe] hydrogenases have been shown to exhibit a high efficiency of H2production, which makes these systems attractive catalysts for renewable energy generating devices. Unfortunately, this class is known for its susceptibility to oxidative degradation, thereby limiting its ability to be used in next‐generation fuel source production devices. In this presentation, we describe the first investigations of the first example of an O2‐tolerant [FeFe] hydrogenase from Clostridium beijerinkii (CbHydA1).1–3. Our FTIR measurements and activity assays show that this enzyme is able to enter an inactivate state under oxidative conditions, is stable for long periods of time in this state, and can be reactivated under reducing conditions. Using FTIR spectroelectrochemistry, we showed that this enzyme is capable of inactivating at unusually low potentials and can inactivate in the absence of oxygen or sulfide, unlike other [FeFe] hydrogenases. Using a combination of FTIR measurements and density functional theory, we are able to propose two possible chemical identities of the inactive state. Finally, we utilized advanced EPR methods to investigate the electronic structure of the active center of CbHydA1, which shed light on the electronic‐structure prerequisites of O2‐tolerance in this unusual [FeFe] hydrogenase.(1) Morra, S.; Arizzi, M.; Valetti, F.; Gilardi, G. Oxygen Stability in the New [FeFe]‐Hydrogenase from Clostridium Beijerinckii SM10 (CbA5H). Biochemistry 2016, 55 (42), 5897–5900. https://doi.org/10.1021/acs.biochem.6b00780.(2) Corrigan, P. S.; Tirsch, J. L.; Silakov, A. Investigation of the Unusual Ability of the [FeFe] Hydrogenase from Clostridium Beijerinckii to Access an O 2 ‐Protected State. J. Am. Chem. Soc. 2020, 142 (28), 12409–12419. https://doi.org/10.1021/jacs.0c04964.(3) Winkler, M.; Duan, J.; Rutz, A.; Felbek, C.; Scholtysek, L.; Lampret, O.; Jaenecke, J.; Apfel, U.‐P.; Gilardi, G.; Valetti, F.; Fourmond, V.; Hofmann, E.; Léger, C.; Happe, T. A Safety Cap Protects Hydrogenase from Oxygen Attack. Nat. Commun. 2021, 12 (1), 756. https://doi.org/10.1038/s41467‐020‐20861‐2.