The unicellular green alga
Chlamydomonas reinhardtii is capable of photosynthetic H
2 production. H
2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H
2ase), the key enzyme responsible for H
2 metabolism in algae, and the Calvin–Benson–Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H
2ase by O
2 coevolved in photosynthesis. Recently, we achieved sustainable H
2 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 H
2ase. Employing membrane-inlet mass spectrometry and
, we now present clear evidence that efficient H
2 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 H
2ase downstream of photosystem I. This occurs exclusively in the absence of CO
2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H
2 photoproduction ceases and instead a slow overall H
2 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) H
2 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 H
2 yield.Many species of green algae have [FeFe]-hydrogenases (H
2ases) (
1) that catalyze the reversible reduction of protons to molecular hydrogen:
[1]Since [FeFe]-H
2ases are extremely O
2-sensitive (
2), reaction 1 typically proceeds under anoxic conditions. With respect to H
2 metabolism,
Chlamydomonas reinhardtii is the most studied alga. This alga possesses two [FeFe]-H
2ases 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 H
2ase via FDX1 (
7). Since [FeFe]-H
2ases 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 H
2 leads to a loss of metabolic energy. In healthy, actively growing
C. reinhardtii cultures, H
2 production is therefore only a temporal phenomenon observed during dark anoxia and upon subsequent onset of illumination (
11). In contrast to dark fermentation, H
2 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 H
2ases (
12,
13). In the light, algae accumulate O
2 that is produced by water oxidation at PSII (
14). As a result, H
2 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 H
2 photoproduction and O
2 evolution, the inhibition of H
2ases by O
2 is frequently quoted as the primary reason for the rapid loss in H
2 photoproduction after the onset of illumination (
17).Alternatively, the loss in the H
2 photoproduction efficiency during illumination could be explained by the light-induced induction of competitive pathways, which may drain reducing equivalents away from the [FeFe]-H
2ase 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 H
2 production has been accumulated in recent studies (
23–
25). As CO
2 fixation provides the strongest sink for photosynthetic reductants, it should play a major role in the cessation of H
2 photoproduction in algae when the CBB cycle is active (
19,
22).For preventing competition between the [FeFe]-H
2ases and the CBB cycle, we recently devised a pulse-illumination protocol that allows H
2 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]-H
2ases. Typically, a train of 1- to 6-s light pulses interrupted by 9-s dark periods is sufficient for sustained H
2 photoproduction in
C. reinhardtii cultures (
23,
25). Our protocol thus differs from earlier pulse-illumination approaches that aimed at preventing the accumulation of O
2 in the cultures (
26).While we could demonstrate competition of [FeFe]-H
2ase with FDPs (
25), the origin of reductants for H
2 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 H
2 and O
2. Although widely proposed in the current literature (
8,
24), the presence of the direct water biophotolysis in H
2-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
with concomitant evolution of
16O
2 and
16,18O
2 during H
2 photoproduction in the pulse-illuminated green alga
C. reinhardtii under anoxic conditions. O
2 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 H
2 photoproduction pathway. Nevertheless, the activity of PSII is indispensable for the sustained process, where it contributes to more than 92% of the final H
2 yield.
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