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

Abstract

Continuous light-dependent H 2 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 0 C is 60 -70 μmol H 2 /mg chlorophyll. At 15 0 C, this reaction proceeds for up to 20 h producing 1O μmol H 2 /mg chlorophyll. O 2 (glucose, glucose oxidase) and peroxide (ethanol, catalase) traps do not extend the lifetime but enhance the rate of H 2 production. Stoichiometry of the coupled system for H 2 production in this system is 2 H 2 formed : 1 glucose consumed. A conversion efficiency of water photolysis for H 2 evolution of 70% was determined from the O 2 produced, measured as the amount of glucose consumed, during the first 2 h of continuous illumination. Cessation of H 2 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 H 2 evolution but longevity of the system remains unchanged. When H 2 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 H 2 production since when H 2 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.