Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae

Abstract

The unicellular green alga <i>Chlamydomonas reinhardtii </i>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 H¹⁸O₂, 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.<br />