A safety cap protects hydrogenase from oxygen attack

Abstract

Abstract[FeFe]-hydrogenases are efficient H2-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic Hox-state and the inactive but oxygen-resistant Hinact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O2-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.

Photochemistry and photoinhibition of the H-cluster of FeFe hydrogenases

“…Regarding oxidative stress, we recently demonstrated that residues that are remote from the active site may change the flexibility of the protein and the reactivity in the 1st coordination sphere of the distal Fe. 89…”

“…The immediate environment of the H-cluster is mostly conserved, although some variations have been observed and may induce unusual catalytic properties 55,85,86 . Non conserved residues that are remote from the active site may This is a post-peer-review, pre-copyedit version of an article published in Sustainable Energy & Fuels (2021) 10.1039/D1SE00756D also affect active site chemistry, as demonstrated using site-directed mutagenesis in the particular case of Clostridium Beijerincki FeFe hydrogenase 89 . They embed a variable number of accessory FeS clusters which are used to mediate long range intramolecular electron transfer: there are 2 accessory clusters in the FeFe hydrogenases from D. desulfuricans (DdHydAB), D. vulgaris (DvHydAB) and M. elsdenii (MeHydA) FeFe hydrogenases, 4 in the FeFe hydrogenases from Clostridium acetobutylicum (CaI), and Clostridium pasteurianum (CpI), and none in the CrHydA1 enzyme from Chlamydomonas reinhardtii (figure 1).…”

Reversible catalysis

“…Chem. doi: 10.1038/s41570-021-00268- 3 (2021) hydrogenases: the active site of NiFe hydrogenases can be over-oxidized into various dead-end species 33 and certain FeFe hydrogenases [34][35][36] also inactivate under moderately oxidizing conditions, which prevents H 2 oxidation and effectively biases the enzyme in the reductive direction. A distinct explanation of the origin of the catalytic bias is related to kinetics 3 .…”

Photosynthetic hydrogen production: Novel protocols, promising engineering approaches and application of semi‐synthetic hydrogenases

“…This conclusion, however, does not necessarily contradict to the well‐known fact of extreme O 2 ‐sensitivity of isolated [FeFe]‐hydrogenase (Stripp et al, 2009). Indeed, the enzyme inside the algal chloroplast could be less susceptible to O 2 inhibition due to a number of different factors such as a change in the catalytic state or in the local environment surrounding [FeFe]‐hydrogenase/its active site (Winkler et al, 2021), enhanced respiration rate, reduced photosynthetic activity, or yet unknown protection mechanisms.…”