Reconstitution of a System for H2 Evolution with Chloroplasts, Ferredoxin, and Hydrogenase

Abstract

Continuous light-dependent H2 production was studied in a reconstituted in vitro system using Spinacea oleracea chloroplasts, Clostridium pasteurianum hydrogenase and Spirulina maxima ferredoxin. PhotosystemII dependent production at 30 0C is 60 -70 μmol H2/mg chlorophyll. At 15 0C, this reaction proceeds for up to 20 h producing 1O μmol H2/mg chlorophyll. O2 (glucose, glucose oxidase) and peroxide (ethanol, catalase) traps do not extend the lifetime but enhance the rate of H2 production. Stoichiometry of the coupled system for H2 production in this system is 2 H2 formed : 1 glucose consumed. A conversion efficiency of water photolysis for H2 evolution of 70% was determined from the O2 produced, measured as the amount of glucose consumed, during the first 2 h of continuous illumination. Cessation of H2 production by the reconstituted system involves inactivation of photosystem II and a limitation in the coupling of low potential electrons to hydrogenase. Increasing ferredoxin leads to more rapid H2 evolution but longevity of the system remains unchanged. When H2 evolution ceases due to inactivation of water-splitting activity of photosystem II, about 40% of the hydro­genase and 25% of photosystem I activity are still present; inactivation is unclear when photo­system I is used to drive H2 production since when H2 production ceases, hydrogenase and photo­system I still retain activity. This may suggest that coupling between low potential reducing equivalents from photosystem I to hydrogenase is impaired.