Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii

[FeFe] hydrogenase from Clostridium beijerinkii (CbHydA1) is an unusual hydrogenase in that it can withstand prolonged exposure to O₂ by reversibly converting into an O₂-protected, inactive state (H_inact). It has been indicated in the past that an atypical conformation of the “SC₃₆₇CP” loop near the [2Fe]_H portion of the six-iron active site (H-cluster) allows the Cys367 residue to adopt an “off-H⁺-pathway” orientation, promoting a facile transition of the cofactor to H_inact. Here, we investigated the electronic structure of the H-cluster in the oxidized state (H_ox) that directly converts to H_inact under oxidizing conditions and the related CO-inhibited state (H_ox-CO). We demonstrate that both states exhibit two distinct forms in electron paramagnetic resonance (EPR) spectroscopy. The ratio between the two forms is pH-dependent but also sensitive to the buffer choice. Our IR and EPR analyses illustrate that the spectral heterogeneity is due to a perturbation of the coordination environment of the H-cluster’s [4Fe4S]_H subcluster without affecting the [2Fe]_H subcluster. Overall, we conclude that the observation of two spectral components per state is evidence of heterogeneity of the environment of the H-cluster likely associated with conformational mobility of the SCCP loop. Such flexibility may allow Cys367 to switch rapidly between off- and on-H⁺-pathway rotamers. Consequently, we believe such structural mobility may be the key to maintaining high enzymatic activity while allowing a facile transition to the O₂-protected state.