The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H₂). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H₂ conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP -reductase (FNR). In the present study, to establish an H₂ photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c₃ (cytc₃) from Desulfovibrio vulgaris. The covalent adduct of cytc₃ and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc₃/PSI complex. The NADPH production by the cytc₃/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H₂ production by the cytc₃/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H₂ production by the hydrogenase/cytc₃/PSI complex was observed (0.30 μmol H₂/mg chlorophyll/h). These results suggest that the cytc₃/PSI complex may produce H₂ in vivo.