344 DANIEL I. ARNON 



The same products of COo assimilation by chlorophyll-free chloroplast 

 extracts, including phosphorylated sugars, were also obtained in a total 

 dark chemosynthesis where TPNHg and ATP were not supplied by a 

 preceding photochemical reaction but were prepared either chemically 

 or enzymically, or were derived from animal material [37]. (Similar 

 experiments were carried out earlier by Racker with a multi-enzyme 

 system that included enzymes from rabbit muscle, yeast, and spinach 

 leaves [40, 41]). 



3. Photosynthetic phosphorylation 



The experiments with isolated chloroplasts have thus underlined the 

 essence of photosynthesis in green plants, i.e. the energy conversion 

 problem, as comprising those chloroplast reactions in which TPNHo and 

 ATP are formed by light. With respect to TPNH., it has already been 

 shown by several laboratories that isolated chloroplasts were capable of 

 reducing TPN to TPNHo in light, with a simultaneous evolution of 

 oxygen [42, 43, 5]. What remained to be determined was the source of 

 ATP in photosynthesis, or more specifically, the cellular site and the 

 mechanism by which ATP is being formed during photosynthesis. From 

 the standpoint of cellular physiology, the important question is whether the 

 ATP used in photosynthesis is supplied by some light-driven assimilation 

 of inorganic phosphate that is peculiar to photosynthesis, or whether the 

 ATP used in photosynthesis is supplied by respiration. 



Before the recent investigations with isolated chloroplasts the only 

 cytoplasmic particles known to form ATP were mitochondria, by the 

 process of oxidative phosphorylation [44]. Oxidative phosphorylation by 

 mitochondria has therefore usually been invoked in explaining the source 

 of ATP used in photosynthesis (see, for example. Fig. 7 in ref. [45] ; also 

 review, ref. [46]). In early models of ATP formation in photosynthesis it 

 was proposed that the reduction of pyridine nucleotide was carried out by 

 illuminated chloroplasts and the resulting reduced pyridine nucleotide was 

 re-oxidized with molecular oxygen by mitochondria [47]. This coupled 

 chloroplast-mitochondrial system differed from conventional oxidative 

 phosphorylation only in the source of the reduced pyridine nucleotide. In 

 one case the pyridine nucleotide was reduced by light and in the other by 

 a respiratory substrate. The phosphorylation reactions proper leading to 

 the synthesis of ATP were in both cases dependent on enzymes localized 

 in mitochondria. 



This model for the generation of ATP in photosynthesis posed a 

 serious difficulty. The most specialized photosynthetic tissue, the meso- 

 phyll of leaves, is noted for its paucity of mitochondria. Within the 

 mesophyll cells, especially in the palisade parenchyma, chloroplasts are the 

 dominant cytoplasmic bodies; mitochondria are few [48, 49]. It was diffi- 



