Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP ⁺ -oxidoreductase (FNR) enzymes in vitro

Abstract

Photosynthetic water splitting, coupled to hydrogenase-catalyzed hydrogen production, is considered a promising clean, renewable source of energy. It is widely accepted that the oxygen sensitivity of hydrogen production, combined with competition between hydrogenases and NADPH-dependent carbon dioxide fixation are the main limitations for its commercialization. Here we provide evidence that, under the anaerobic conditions that support hydrogen production, there is a significant loss of photosynthetic electrons toward NADPH production in vitro. To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP þ -oxidoreductase (FNR), and NADP þ . Replacing the hydrogenase with a ferredoxin-hydrogenase fusion switched the bias of electron transfer from FNR to hydrogenase and resulted in an increased rate of hydrogen photoproduction. These results suggest a new direction for improvement of biohydrogen production and a means to further resolve the mechanisms that control partitioning of photosynthetic electron transport.

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…2), whose presence should decrease and increase the amplitude of the proton gradient, respectively. In this respect, it was recently shown that NADPH reduction by FNR prevents an efficient H 2 production by HYDA in vitro (Yacoby et al, 2011). This might be due to the low affinity of HYDA for FDX (K m = 35 mM; Happe and Naber, 1993), close to two orders of magnitude lower than the affinity of FNR for FDX (K m = 0.4 mM; Jacquot et al, 1997).…”

“…At the onset of illumination, only a hydrogenase-dependent LEF occurs (10 s), followed by the induction of PGRL1-dependent PSI-CEF and later the CBB cycle (120-240 s). When the CBB cycle has been activated by ATP, the NADP + pool is partially oxidized and FNR, being more efficient in competing for reduced FDX than hydrogenases (see next section; Yacoby et al, 2011), rapidly drives the entire electron flux toward CO 2 reduction (more than 360 s). In single mutants, increase in CBB cycle activity is only slightly delayed (Fig.…”

“…In this respect, at least three hypotheses have been formulated: (1) the inhibition of hydrogenase by O 2 produced by water photolysis (Ghirardi et al, 1997;Cohen et al, 2005), (2) the competition between ferredoxin-NADP + oxidoreductase (FNR) and hydrogenase activity for reduced FDX (Yacoby et al, 2011), and (3) the inhibition of electron supply to hydrogenases by the proton gradient generated by another AEF, the cyclic electron flow around PSI (PSI-CEF; Tolleter et al, 2011). First described by Arnon (1955), PSI-CEF consists in a reinjection of electrons from reduced FDX or NADPH pool in the plastoquinone (PQ) pool.…”

Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii

“…We prepared in vitro reactions using Chlamydomonas thylakoid membranes, which harbor membrane-associated FNR activity (42). FDX1 mediated the highest rates of NADPH photo-production (7.896 mol of NADPH h Ϫ1 mg of Chl…”

“…Reactions using isolated Chlamydomonas thylakoid membranes (42) were prepared by adding purified recombinant HYDA1 (34) and purified recombinant FDX (see "Experimental Procedures"). We found that FDX1 drives the highest rates of hydrogen photo-production (0.52 nmol of H 2 h Ϫ1 ), although FDX2-mediated hydrogen photoproduction was 3-fold lower (0.18 nmol of H 2 h Ϫ1 ) (Fig.…”