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

Continuous light-dependent H₂ production was studied in a reconstituted in vitro system using Spinacea oleracea chloroplasts, Clostridium pasteurianum hydrogenase and Spirulina maxima ferredoxin. Photosystem II-dependent production at 30 °C is 60-70 μmol H₂/mg chlorophyll. At 15 °C, this reaction proceeds for up to 20 h producing 10 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.