Engineering O₂-Tolerant Chimeric Hydrogenases Optimized for Ferredoxin Coupling in Synechocystis 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 O₂-tolerant SH of Cupriavidus necator (CnSH) and the ferredoxin-interacting SH of Synechocystis sp. PCC6803 (SynSH). By engineering chimeric HoxU and HoxF subunits, we developed constructs─MixSH, Ch-HoxEF_Syn+U_Cn, and Ch-HoxU^swapCTD─that successfully couple the CnHoxYH hydrogenase module to the SynHoxEFU reductase module while retaining O₂ tolerance and enhancing interaction with reduced ferredoxin. The lithoautotrophic growth of C. necator confirmed the tolerance of these variants to O₂, while activity assays in Synechocystis demonstrated partial hydrogenase function, including H₂ consumption and fermentative H₂ 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 CnHoxF and identified the CnHoxF N-terminal domain as structurally and functionally analogous to SynHoxE. Although electron transfer efficiency and activity in Synechocystis remained limited, this work validates the modular engineering of [NiFe]-hydrogenases, uniting O₂-tolerance with ferredoxin interaction and offering a foundational step toward photosynthesis-coupled H₂ production.