Stepwise generation of the natural oxidant in a reconstituted chloroplast system

Isolated chloroplasts which have lost their envelopes and, in consequence, the soluble components which constitute the stroma, will nevertheless evolve O(2) when supplied with an artificial oxidant (the Hill reaction). They will also evolve O(2) with NADP as the Hill oxidant if supplemented with ferredoxin. With catalytic NADP, continuing O(2) evolution can be maintained by the inclusion of a suitable reaction or reaction sequence which reoxidizes NADPH.In the Benson-Calvin cycle the terminal oxidant is glycerate 1,3-bisphosphate which is generated by phosphorylation of 3-phosphoglycerate, its immediate precursor. Experiments with a reconstituted chloroplast system are described in which this reaction sequence is catalyzed by stromal protein and supported by photophosphorylation of catalytic ADP. In the presence of CO(2), 3-phosphoglycerate can be progressively replaced by ribulose 1,5-bisphosphate, ribose 5-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, and finally by glyceraldehyde 3-phosphate. In this last instance the natural oxidant is regenerated from its own reduction product (via the carboxylation step) and the reaction sequence therefore involves the entire photosynthetic carbon cycle.