Engineering O ₂ -Tolerant Chimeric Hydrogenases Optimized for Ferredoxin Coupling in <i>Synechocystis</i> sp. PCC 6803

The development of hydrogenases capable of operating under oxygenic photosynthetic conditions remains a key challenge for sustainable biohydrogen production. In this study, we developed a series of chimeric NAD + -reducing [NiFe]-hydrogenases (SH) combining structural elements from the O 2 -tolerant SH of Cupriavidus necator ( Cn SH) and the ferredoxin-interacting SH of Synechocystis sp. PCC6803 ( Syn SH). By engineering chimeric HoxU and HoxF subunits, we developed constructs─MixSH, Ch-HoxEF Syn +U Cn, and Ch-HoxU swapCTD ─that successfully couple the Cn HoxYH hydrogenase module to the Syn HoxEFU reductase module while retaining O 2 tolerance and enhancing interaction with reduced ferredoxin. The lithoautotrophic growth of C. necator confirmed the tolerance of these variants to O 2, while activity assays in Synechocystis demonstrated partial hydrogenase function, including H 2 consumption and fermentative H 2 production. Notably, Ch-HoxEF Syn +U Cn retained ferredoxin interaction despite lacking the [4Fe4S] U4 cluster, showing [2Fe2S] F2 in HoxF as a functional ferredoxin-binding site. Moreover, we achieved the artificial integration of a [2Fe2S] cluster into Cn HoxF and identified the Cn HoxF N-terminal domain as structurally and functionally analogous to Syn HoxE. Although electron transfer efficiency and activity in Synechocystis remained limited, this work validates the modular engineering of [NiFe]-hydrogenases, uniting O 2 -tolerance with ferredoxin interaction and offering a foundational step toward photosynthesis-coupled H 2 production.