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A Dynamic Water Channel Affects O 2 Stability in [FeFe]-Hydrogenases.

Authors :: Brocks C
Das CK
Duan J
Yadav S
Apfel UP
Ghosh S
Hofmann E
Winkler M
Engelbrecht V
Schäfer LV
Happe T
Source :: ChemSusChem [ChemSusChem] 2024 Feb 08; Vol. 17 (3), pp. e202301365. Date of Electronic Publication: 2023 Nov 17.
Publication Year :: 2024
Abstract: [FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O <subscript>2</subscript> ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4Fe <subscript>H</subscript> ) and a unique diiron moiety (2Fe <subscript>H</subscript> ). Previous attempts to prevent O <subscript>2</subscript> -induced damage have focused on enhancing the protein's sieving effect for O <subscript>2</subscript> by blocking the hydrophobic gas channels that connect the protein surface and the 2Fe <subscript>H</subscript> . In this study, we aimed to block an O <subscript>2</subscript> diffusion pathway and shield 4Fe <subscript>H</subscript> instead. Molecular dynamics (MD) simulations identified a novel water channel (W <subscript>H</subscript> ) surrounding the H-cluster. As this hydrophilic path may be accessible for O <subscript>2</subscript> molecules we applied site-directed mutagenesis targeting amino acids along W <subscript>H</subscript> in proximity to 4Fe <subscript>H</subscript> to block O <subscript>2</subscript> diffusion. Protein film electrochemistry experiments demonstrate increased O <subscript>2</subscript> stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O <subscript>2</subscript> in the environment of the 4Fe <subscript>H</subscript> in both cases. The results strongly suggest that, in wild type proteins, O <subscript>2</subscript> diffuses from the 4Fe <subscript>H</subscript> to the 2Fe <subscript>H</subscript> . These results reveal new strategies for improving the O <subscript>2</subscript> stability of [FeFe]-hydrogenases by focusing on the O <subscript>2</subscript> diffusion network near the active site.<br /> (© 2023 The Authors. ChemSusChem published by Wiley-VCH GmbH.)