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 O₂-tolerant SH of <i>Cupriavidus necator</i> (<i>Cn</i>SH) and the ferredoxin-interacting SH of <i>Synechocystis</i> sp. PCC6803 (<i>Syn</i>SH). By engineering chimeric HoxU and HoxF subunits, we developed constructs─MixSH, Ch-HoxEF_<i>Syn</i>+U_<i>Cn</i>, and Ch-HoxU^swapCTD─that successfully couple the <i>Cn</i>HoxYH hydrogenase module to the <i>Syn</i>HoxEFU reductase module while retaining O₂ tolerance and enhancing interaction with reduced ferredoxin. The lithoautotrophic growth of <i>C. necator</i> confirmed the tolerance of these variants to O₂, while activity assays in <i>Synechocystis</i> demonstrated partial hydrogenase function, including H₂ consumption and fermentative H₂ production. Notably, Ch-HoxEF_<i>Syn</i>+U_<i>Cn</i> 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 <i>Cn</i>HoxF and identified the <i>Cn</i>HoxF N-terminal domain as structurally and functionally analogous to <i>Syn</i>HoxE. Although electron transfer efficiency and activity in <i>Synechocystis</i> 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.