Light-induced H 2 generation in a photosystem I-O 2 -tolerant [FeFe] hydrogenase nanoconstruct.

The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H 2 ). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O 2 sensitivity, binding specificity, or H 2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O 2 -tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS] 4 linker group ( Cb HydA1-PsaE). We demonstrate that the Cb HydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H 2 at a rate of 84.9 ± 3.1 µmol H 2 mg chl -1 h -1 . Further, when prepared and illuminated in the presence of O 2 , the nanoconstruct retains the ability to generate H 2 , though at a diminished rate of 2.2 ± 0.5 µmol H 2 mg chl -1 h -1 . This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O 2 -tolerant [FeFe] hydrogenase opens the possibility for an in vivo H 2 generating system that can function in the presence of O 2 .