Tuning Electron Transfer Pathway in [NiFe]-Hydrogenase via Silver Nanocluster-Directed Electron Injection

[NiFe]-hydrogenases mediate reversible H2/proton interconversion through a relay of three iron–sulfur (FeS) clusters. Here, we report an electron transfer (ET) rewiring strategy in Escherichia coli hydrogenase-2 (Hyd-2) by engineering surface cysteine mutations (E261C and V137C) to enable site-selective conjugation with silver nanoclusters (AgNC). This approach bypasses the distal [4Fe-4S] cluster and directs photoelectrons into either the medial [3Fe-4S] or the proximal [4Fe-4S] cluster. Under visible-light irradiation, the E261C-3S-Hyd-2 variant exhibited a 1.5-fold higher H2 evolution rate compared with V137C-3S-Hyd-2, despite the longer ET pathway. Combined Fourier transform infrared spectroscopy, protein film electrochemistry, and impedance analyses revealed that the medial [3Fe-4S] cluster acts as a redox-active relay element, lowering reorganization energy and enhancing ET efficiency, in agreement with Marcus theory. These findings demonstrate that FeS-cluster topology can override simple distance constraints, highlighting the critical role of intermediate cofactors in optimizing the electron flux. This work not only establishes a generalizable strategy for probing and controlling enzymatic ET pathways but also provides design principles for constructing efficient biohybrid and bioinspired catalysts.