Competition for electrons between FNR and the fused Fd-15aa-HydA. (A) Both hydrogen and NADPH production rates were measured under competitive and noncompetitive conditions. Each in vitro reaction contained plant thylakoids (25 μg chlorophyll, Chl) in combination with purified HydA (100 nM) or Fd-15aa-HydA (1 μM) and increasing concentrations of Fd up to 40 μM (x axis). Hydrogen production rates are shown (Upper) (noncompetitive, in the absence of NADP+; competitive, in the presence of NADP+). A significant increase in hydrogen production rates occurs under competitive conditions when HydA is replaced with Fd-15aa-HydA (brown versus green plots, respectively). NADPH production rates are shown (Lower) (noncompetitive, in the absence of HydA; competitive, in the presence of HydA or Fd-15aa-HydA). The NADPH production rates decrease, but are not completely inhibited, by the addition of Fd-15aa-HydA (green plot), whereas the rates increase with addition of HydA (brown plot) as observed before (see main text for discussion). This result strongly suggests that electrons are diverted at PS-I from FNR to Fd-15aa-HydA, resulting in increased hydrogen production rates but decreased NADPH production rates. (D) The schematic shows that, in the absence of free Fd, electrons generated at PSI during illumination are directly transferred to Fd-15aa-HydA (mode 1) and photohydrogen production. A small fraction (indicated by the small black arrow) of electrons are transferred from Fd of Fd-15aa-HydA to FNR for NADPH production. (C) The pie chart summarizes the data that are modeled in D. In the presence of Fd-15aa-HydA, and in the absence of free Fd, only approximately 10% of photosynthetic electrons are allocated to FNR and NADPH production and the remaining 90% to hydrogen production. (B) The schematic shows that both Fd-15aa-HydA (mode 1) and Fd (mode 2) are directly reduced at PSI to support hydrogen production, or simultaneous NADPH and hydrogen production (mode 2). The two electron-transfer modes at PSI allow for hydrogen production to compete with NADPH production. (E) The pie chart summarizes the results from A for 10 μM Fd, showing that Fd-15aa-HydA successfully competes with FNR to divert 60–70% of the electrons generated at PSI in thylakoids towards hydrogen production. In contrast, HydA not only catalyzes very low rates of hydrogen production in the presence of FNR, but also stimulates NADPH production rates (see also A and SI Text).