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

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 H218O, 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.

Many species of green algae have [FeFe]-hydrogenases (H₂ases) (1) that catalyze the reversible reduction of protons to molecular hydrogen:2H++2e−⇄H2.[1]Since [FeFe]-H₂ases are extremely O₂-sensitive (2), reaction 1 typically proceeds under anoxic conditions. With respect to H₂ metabolism, Chlamydomonas reinhardtii is the most studied alga. This alga possesses two [FeFe]-H₂ases in the chloroplast, HYDA1 and HYDA2 (3, 4). In the light, they accept electrons from photosynthetically reduced ferredoxin (FDX1) (5), while in the dark electrons come from the activity of pyruvate ferredoxin oxidoreductase (PFR1) (6). PFR1 catalyzes the oxidation of pyruvate to acetyl-CoA, and its activity is linked to H₂ase via FDX1 (7). Since [FeFe]-H₂ases interact with the photosynthetic electron transport chain at the level of ferredoxin, they may accept electrons originating both from water oxidation via the photosystem II (PSII)-dependent pathway (“direct water biophotolysis”) and from the degradation of organic substrates via a PSII-independent mechanism (“indirect water biophotolysis” or “indirect pathway”) (8). In the latter case, the reductants are supplied to the plastoquinone (PQ) pool by the type II NADPH dehydrogenase (NDA2), thus bypassing PSII (9, 10).The release of H₂ leads to a loss of metabolic energy. In healthy, actively growing C. reinhardtii cultures, H₂ production is therefore only a temporal phenomenon observed during dark anoxia and upon subsequent onset of illumination (11). In contrast to dark fermentation, H₂ photoproduction is a very efficient process that proceeds for only a short period of time (from a few seconds to a few minutes). Two theories have been developed to explain the short duration. The first is based on the oxygen sensitivity of H₂ases (12, 13). In the light, algae accumulate O₂ that is produced by water oxidation at PSII (14). As a result, H₂ photoproduction may cease over time (14, 15), and the duration of this process is reported to shorten with increased light intensity (16). Because of the negative correlation between the rates of H₂ photoproduction and O₂ evolution, the inhibition of H₂ases by O₂ is frequently quoted as the primary reason for the rapid loss in H₂ photoproduction after the onset of illumination (17).Alternatively, the loss in the H₂ photoproduction efficiency during illumination could be explained by the light-induced induction of competitive pathways, which may drain reducing equivalents away from the [FeFe]-H₂ase enzyme (18, 19). Candidates for this role are the Mehler-like reaction driven by flavodiiron proteins (FDPs) (15, 20, 21) and the Calvin–Benson–Bassham (CBB) cycle (22). Compelling evidence for the competition between these two pathways and H₂ production has been accumulated in recent studies (23–25). As CO₂ fixation provides the strongest sink for photosynthetic reductants, it should play a major role in the cessation of H₂ photoproduction in algae when the CBB cycle is active (19, 22).For preventing competition between the [FeFe]-H₂ases and the CBB cycle, we recently devised a pulse-illumination protocol that allows H₂ production in nutrient-replete algal cultures for up to 3 d (23). To achieve this, we specifically selected the duration of light pulses in the light/dark sequence to avoid activation of the CBB cycle, thus allowing for the redirection of photosynthetic electrons toward the [FeFe]-H₂ases. Typically, a train of 1- to 6-s light pulses interrupted by 9-s dark periods is sufficient for sustained H₂ photoproduction in C. reinhardtii cultures (23, 25). Our protocol thus differs from earlier pulse-illumination approaches that aimed at preventing the accumulation of O₂ in the cultures (26).While we could demonstrate competition of [FeFe]-H₂ase with FDPs (25), the origin of reductants for H₂ photoproduction in the pulse-illuminated algae remained unclear. The relatively high efficiency of the process suggests the involvement of water oxidation by PSII, and consequently the simultaneous production of H₂ and O₂. Although widely proposed in the current literature (8, 24), the presence of the direct water biophotolysis in H₂-producing green algae has not yet been proven by direct experimental data.In the present study, we provide clear evidence for the presence of PSII-dependent oxidation of ¹⁸O-labeled water H218O with concomitant evolution of ¹⁶O₂ and ^16,18O₂ during H₂ photoproduction in the pulse-illuminated green alga C. reinhardtii under anoxic conditions. O₂ evolution is balanced by light-dependent and light-independent respiration that sustains the anoxic condition. We also demonstrate that the loss of PSII activity in algae can be partly compensated by the PSII-independent H₂ photoproduction pathway. Nevertheless, the activity of PSII is indispensable for the sustained process, where it contributes to more than 92% of the final H₂ yield.