However, the effect of expressing O2 sequesters, such as leghemoglobin and the pyruvate oxidase enzyme, in Chlamydomonas should be analyzed more carefully to determine (a) the total O2-binding capability of leghemoglobin molecules, and how
the O2 is eventually released to the medium, and (b) the efficacy of the pyruvate oxidase reaction in long-term, high-H2-producing conditions. An additional approach under consideration HDAC inhibitors list involves the expression of one of the clostridial [FeFe]-hydrogenases in Chlamydomonas. These enzymes have been shown to have two orders of magnitude higher tolerance to O2 in vitro, and one needs to verify whether it maintains its higher O2 tolerance when physiologically connected to the Chlamydomonas photosynthetic apparatus as well. Barrier: proton gradient The downregulation of photosynthetic LEF by non-dissipation of the proton gradient in H2-producing cell was addressed by isolation of a mutant
deficient in PGRL1, as described in “Non-dissipated proton gradient and state transitions” sections. The PGRL1 protein is a component of a supercomplex that includes PSI-LHCI-LHCII-FNR-Cytochrome b6/f; this supercomplex is proposed to mediate CEF, and its operation is induced by high light conditions. When PGRL1 is genetically disrupted, the CEF around PSI becomes non-operational (Tolleter et al. 2011). The pgrl1 mutant strain was shown to exhibit lower CEF GANT61 nmr and increased hydrogen production under both short-term (argon-induced) and long-term (sulfur-deprivation-induced) anaerobiosis under high light. The authors concluded that the proton gradient generated by CEF in WT cells under high illumination strongly limits the electron supply to hydrogenase,
and it can be overcome by disrupting components of the supercomplex. Moreover, as expected, the mutant strain exhibited reduced NPQ, likely resulting from the decrease in the CEF-dependent proton gradient. Although it has been shown recently that state transitions do not control CET (Lucker and Kramer 2013; Blebbistatin order Takahashi et al. 2013), a mutant blocked in state 1 (stm6) showed no CET, higher respiratory metabolism, large starch reserves, second and a low dissolved O2 concentration (40 % of the wild type (WT)), resulting in increased hydrogen production following anaerobic induction. No direct effect on PSII activity was reported, possibly due to the fact that anaerobiosis could be achieved faster—thus protecting PSII from irreversible photoinhibition. The H2-production rates of were 5–13 times higher than the control WT strain over a range of conditions (light intensity, culture time, and addition of uncouplers). More recent studies demonstrated that most PSII centers are “closed” in the stm6 mutant during the anaerobic phase, and that, under sulfur-deprivation conditions, water splitting by the remaining open PSII supplies the majority of electrons for H2 synthesis (Volgusheva et al. 2013).