Fig. 2.

Kinetics of light-driven NADPH production versus hydrogen production. Each of the in vitro reactions contained either plant thylakoids (25 μg chlorophyll, Chl) (black or green) or purified plant PSI (10 μg Chl) (blue and red) in combination with purified HydA and Fd to measure hydrogen production either in a noncompetitive reaction (the absence of NADP⁺ for thylakoids, or with NADP⁺ but heat inactivated FNR for PSI reactions) or competitive conditions (both active FNR and NADP⁺). The competition between FNR and HydA for Fd was tested in reactions containing isolated thylakoids or purified PSI with varying concentrations of Fd (A and D), HydA (B), and FNR (C). (A) The rates of hydrogen production as a function of Fd concentration. HydA (100 nM) was incubated with (competitive) or without (noncompetitive) NADP⁺ (2.5 mM) and active FNR (copurified with thylakoids, or added at 50 nM to purified PSI). Competition with NADPH production resulted in approximately 75% inhibition of hydrogen production at Fd concentrations higher than 5 μM with both thylakoids and PSI. (B) The rates of hydrogen production as a function of HydA concentration. HydA was added at increasing concentrations up to 850 nM for thylakoids or up to 200 nM for PSI as the concentrations above 100 nM are saturating. (C) The rates of NADPH production as a function of FNR concentration. The addition of external FNR to thylakoids did not alter the NADPH production rate, indicating this pathway is saturated by a membrane-associated FNR. The addition of HydA at a concentration of 100 nM, together with 10 μM Fd had no effect on either thylakoid or PSI mediated NADPH production rates. (D) The rates of NADPH production as a function of Fd concentration. FNR that either copurified with thylakoids or was exogenously added to PSI (50 nM) was combined in reactions Fd at up to 40 μM.