Improved O 2 ‐tolerance in variants of a H 2 ‐evolving [NiFe]‐hydrogenase from Klebsiella oxytoca HP1

In this study, we investigated the mechanism of O 2 tolerance of Klebsiella oxytoca HP1 H 2 ‐evolving hydrogenase 3 (KHyd3) by mutational analysis and three‐dimensional structure modeling. Results revealed that certain surface amino acid residues of KHyd3 large subunit, in particular those at the outer entrance of the gas channel, have a visible effect on its oxygen tolerance. Additionally, solution pH, immobilization and O 2 partial pressure also affect KHyd3 O 2 ‐tolerance to some extent. We propose that the extent of KHyd3 O 2 ‐tolerance is determined by a balance between the rate of O 2 access to the active center through gas channels and the deoxidation rate of the oxidized active center. Based on our findings, two higher O 2 ‐tolerant KHyd3 mutations G300E and G300M were developed.