M. B. Allen

Photosynthesis by isolated chloroplasts. IV. General concept and comparison of three photochemical reactions.

Abstract 1. 1. Procedures are described for the preparation of chloroplasts capable of carrying out three photochemical reactions, each representing an increasingly complex phase of photosynthesis: photolysis of water (Hill reaction), esterification of inorganic phosphate into adenosine triphosphate (photosynthetic phosphorylation) and the reduction of carbon dioxide to the level of carbohydrates with a simultaneous evolution of oxygen. 2. 2. The three photochemical reactions were separable by variations in the technique for preparation of chloroplasts and by differential inhibition by several reagents. Inhibition of a more complex phase of photosynthesis does not affect the similar one which precedes it and, conversely, the inhibition of a simpler phase of photosynthesis is paralled by an inhibition of the more complex phase which follows. 3. 3. Reversible inhibition of CO 2 fixation and photosynthetic phosphorylation, but not of photolysis, by sulfhydryl group inhibitors suggests that sulfhydryl compounds (enzymes, cofactors, or both) are involved in phosphorylation and CO 2 fixation, but not in the primary conversion of light into chemical energy as measured by the Hill reaction. 4. 4. Evidence is presented in support of the conclusion that the synthesis of ATP by green cells occurs at two distinct sites: anaerobically in chloroplasts, by photosynthetic phosphorylation, and aerobically in smaller cytoplasmic particles, presumably mitochondria, by oxidative phosphorylation independent of light. 5. 5. A general scheme of photosynthesis by chloroplasts, consistent with these findings, is presented.

Photosynthesis by isolated chloroplasts V. Phosphorylation and carbon dioxide fixation by broken chloroplasts

Abstract 1. 1. Photosynthetic esterification of inorganic phosphate into adenosine triphosphate, and reduction of CO 2 to the level of carbohydrate, hitherto found to occur only in whole chloroplasts, have now been observed with chloroplasts broken by treatment with water. 2. 2. Broken chloroplasts retained only two of the three groups of enzymes contained in whole chloroplasts, namely, those controlling the photolysis of water and photosynthetic phosphorylation. At least some of the enzymes concerned in reduction of CO 2 were leached out by treating the chloroplasts with water, with the result that CO 2 fixation was completely abolished. 3. 3. On addition of the requisite cofactors, the capacity of broken chloroplasts for photosynthetic phosphorylation was the same as that of whole chloroplasts. 4. 4. The restoration to broken chloroplasts of the full capacity for photosynthetic CO 2 fixation of whole chloroplasts required the addition of pyridine nucleotides, adenosine triphosphate, and the soluble enzymes removed by water treatment of whole chloroplasts. 5. 5. An additional several-fold increase in the rate of CO 2 fixation by the reconstituted broken chloroplast system was obtained by the further addition of one of several compounds, principally phosphorylated sugars. This has resulted in a level of CO 2 fixation by broken chloroplasts which is much higher than that obtained with whole chloroplasts.

Photosynthesis by isolated chloroplasts: VII. Vitamin K and riboflavin phosphate as cofactors of cyclic photophosphorylation☆

Abstract Vitamin K substances and FMN appear to catalyze separate pathways of cyclic photophosphorylation. The FMN pathway shows a dependence on added TPN and greater sensitivity to inhibition by dinitrophenol and o -phenanthroline than the vitamin K pathway. Both pathways are inhibited by p -chloromercuribenzoate, gramicidin and methylene blue but not by arsenite or antimycin A. Cyclic photophosphorylation catalyzed by phenazine methosulfate resembled the vitamin K pathway in its independence from added TPN and resistance to inhibition by dinitrophenol and o -phenanthroline. The role of vitamin K in phosphorylations by plant and animal tissues is reviewed. A possible physiological role for cyclic photophosphorylation in photosynthesis of green plants is suggested.

Photosynthesis by isolated chloroplasts: VIII. Photosynthetic phosphorylation and the generation of assimilatory power

Abstract Photochemical ATP formation by isolated chloroplasts was coupled with a reduction of ferricyanide or TPN. Esterification of two moles of orthophoshate was coupled with the formation of two moles of TPNH 2 and the evolution of one mole of oxygen. The addition of catalytic amounts of FMN, vitamin K or phenazine methosulfate to the TPN phosphorylating system suppressed TPNH 2 accumulation as well as oxygen evolution and greatly increased the light-dependent ATP formation. A revised general scheme is presented for photosynthesis by isolated chloroplasts.

Photosynthesis by isolated chloroplasts. VI. Rates of conversion of light into chemical energy in photosynthetic phosphorylation.

Abstract Under improved experimental conditions, rates of photosynthetic phosphorylation up to 500 micromoles of orthophosphate esterified per hour per mg of chlorophyll were obtained with isolated chloroplast fragments. Expressed on a nitrogen basis these rates correspond to about 780 micromoles of orthophosphate esterified per hour per mg N. These rates are similar to maximum rates of photosynthesis in intact leaves of land plants with optimal light and CO 2 supply (about 180 micromoles of CO 2 fixed per hour per mg of chlorophyll) and are several times higher than rates of oxidative phosphorylation from mitochondria from various plant and animal sources. The significance of these rates in the evaluation of the role of photosynthetic phosphorylation as a source of ATP in photosynthesis is discussed.