Water oxidation by photosystem II is the primary source of electrons for sustained H₂ photoproduction in...

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H₂ production. H₂ evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H₂ase), the key enzyme responsible for H₂ metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H₂ase by O₂ coevolved in photosynthesis. Recently, we achieved sustainable H₂ photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H₂ase. Employing membrane-inlet mass spectrometry and [Formula: see text], we now present clear evidence that efficient H₂ photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H₂ase downstream of photosystem I. This occurs exclusively in the absence of CO₂ fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H₂ photoproduction ceases and instead a slow overall H₂ uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H₂ photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H₂ yield.