VOL. 48, 1962 BIOCHEMISTRY: HALL AND ARNON 833 10 De Robertis, E., J. Biophy8. Biochem. Cytol., 2, 319 (1956). 11 Eakin, R. M., and J. A. Westfall, ibid., 8, 483 (1960). 12 Eakin, R. M., these PROCEEDINGS, 47, 1084 (1961). 13 Wolken, J. J., in The Structure of the Eye, ed. G. K. Smelser (New York: Academic Press, 1961). 14 Wald, G., ibid. 16 Eakin, R. M., and J. A. Westfall, Embryologia, 6, 84 (1961). 16 Sj6strand, F. S., in The Structure of the Eye, ed. G. K. Smelser (New York: Academic Press, 1961). 17 Miller, W. H., in The Cell, ed. J. Brachet and A. E. Mirsky (New York: Academic Press, 1960), part IV. 18 Miller, W. H., J. Biophys. Biochem. Cytol., 4, 227 (1958). 19 Hesse, R., Z. wiss. Zool., 61, 393 (1896). 20 Bradke, D. L., personal communication. 21 Wolken, J. J., Ann. N. Y. Acad. Sci., 74, 164 (1958). 22 Rohlich, P., and L. J. Torok, Z. wiss. Zool., 54, 362 (1961). 28 Eakin, R. M., and J. A. Westfall, J. Ultrastr. Res. (in press). 24 Franz, V., Jena. Z. Naturwiss., 59, 401 (1923). PHOTOSYNTHETIC PHOSPHORYLATION ABOVE AND BELOW 00C BY DAVID 0. HALL* AND DANIEL I. ARNONt DEPARTMENT OF CELL PHYSIOLOGY, UNIVERSITY OF CALIFORNIA, BERKELEY Read before the Academy, April 25, 1962 CO2 assimilation in photosynthesis consists of a series of dark enzymatic reactions that are driven solely by adenosine triphosphate and reduced pyridine nucleotide. 1-4 The same reactions are now known to operate in nonphotosynthetic cells.5-"3 It follows, therefore, that the distinction between carbon assimilation in photosyn- thetic and nonphotosynthetic cells lies in the manner in which ATP14 and PNH214 are formed. Photosynthetic cells form ATP and PNH2 at the expense of radiant energy, whereas nonphotosynthetic cells form them at the expense of energy re- leased by dark chemical reactions. Thus, the understanding at the biochemical level of the key event in photo- synthesis, the conversion of radiant energy into physiologically useful chemical energy, depends on the elucidation of the mechanisms of the photochemical reactions that form ATP and PNH2. These reactions, two in number, are identified in the current nomenclature'5 as cyclic (equation (1)) and noncyclic photophosphorylation (equation (2)): light ADP + P- ATP (1) cofactor light ADP + P + TPN + 2H+ + 20H- -> ATP + TPNH2 + H20 + 1/202. (2) Both reactions were first found in isolated chloroplasts16-'8 but are now known to occur in representatives of all the major groups of photosynthetic organisms and are, therefore, considered of general importance in photosynthesis. Cyclic19 and a "bacterial type" of noncyclic2O photophosphorylation were found in chromatophores Downloaded by guest on October 1, 2021 834 BIOCHEMISTRY: HALL AND ARNON PROC. N. A. S. of photosynthetic bacteria; cyclic photophosphorylation has also been found in cell-free preparations of algae.21' 22 (For a more complete review of literature see ref. 23 and 23a.) Cyclic and noncyclic photophosphorylation have been localized in subcellular photosynthetic particles (chloroplasts and chromatophores) which, under proper conditions, remain functional after isolation from the cell. This has made it possi- ble to separate physically the light-induced formation of ATP (and PNH2) in chloroplasts from ATP formation by oxidative phosphorylation in mitochondria. In the intact cell these two processes occur concurrently. The use of isolated photo- synthetic particles and their later fractionation was also important in lowering, or removing altogether, cellular permeability barriers to the entry of such key inter- mediates as ADP and PN, the addition of which paved the way for the discovery of the photophosphorylation reactions and the study of their mechanisms. Investigations of cyclic and noncyclic photophosphorylation have led to the formulation of "electron flow" reaction mechanisms" which envisage the same TABLE 1 CYCLIC AND NONCYCLIC PHOTOPHOSPHORYLATION AT -80C ATP formed System (#moles) Cyclic PMS 10.5 Vit. K3 4.5 FMN 4.9 PMS, dark 0.7 PMS, ADP omitted 0.2 Noncyclic TPN 4.2 FeCy 4.1 No cofactor added 1.0 Reaction was run for 10 minutes at an illumination of 50,000 lux. Reaction vessels were continuously flushed with purified nitrogen gas. The reaction mixture included, in a final volume of 3 ml, chloroplast fragments (Pis) contain- ing 2.0 mg chlorophyll, 0.3 ml methanol, and the following in pmoles: Tris/ acetate buffer, pH 8.0, 80; MgSOi, 10- KsHP204 15; and ADP, 15. The following were added (in pmoles) where indicated: 1iMS, 0.4; vitamin Ks, 0.3; FMN, 0.3; KsFe(CN)e' 15' and TPN, 6. Sodium ascorbate (5 umoles) was added to the FMN and vit. i reaction mixtures; purified TPN-reductase from spinach leaves was added together with TPN. primary physical steps of photon capture by chlorophyll which are subsequently linked to either shorter or longer chemical pathways 'coupled with ATP formation (cf. review2). Cyclic photophosphorylation catalyzed by phenazine methosulfate (methyl phenazonium methosulfate) is viewed as having a shorter chemical path- way24 for ATP formation than cyclic photophosphorylation catalyzed either, by vitamin K3 (menadione) or flavin mononucleotide, and also shorter than noncyclic photophosphorylation. (Compare Figs. 4 and 5 in ref. 23 and Fig. 1 in ref. 25.) The validity of this concept has now been further strengthened by experiments which tested the idea that the shortest chemical pathway for ATP formation may be the least sensitive to temperature. Variants of cyclic and noncyclic photo- phosphorylation in isolated chloroplasts were investigated over a range of tempera- ture from - 100 to 15'C. The results of these experiments reported in this com- munication -have revealed an appreciable light-induced ATP formation below 0C which, under certain conditions, is independent of temperature in the range -10° to 15°0. Downloaded by guest on October 1, 2021 VOL. 48, 1962 BIOCHEMISTRY: HALL, AND ARNON 835 -80C 85 /20 4Q000 Lux 7 f os 0 ck 5 -I~ ~ ~ ~ b80- 4- ~~~~~~~60- VitAkj * ~~~~~40 2 AFeCy C / ~~~~~~~~20 C' 25,000) 50,000 1.0 2.0 light intensify (Lux) m~g ch/c*odiyll FIG. 2.-Cyclic photophosphoryl- FIG. 1.-Effect of light intensity ation (PMS system) at -8O and on cyclic and noncyclic photophos- 150C as a function of chlorophyll phorylation at -80C. The reac- concentration. The reaction mix- tion mixture included in a final vol- ture was the same as described for ume of 3 ml, chloroplast fragments Figure 1 except that, at 150C, 0.3 (P18) containing 2.0 mg chlorophyll, Mmole PMS was added and that the 0.3 ml methanol and the following in chlorophyll concentration was varied smoles: Tris/acetate buffer, pH as indicated. Illumination was 8.0, 80; MgSO4, 10; ADP, 15; 40,000 lux. K2HP3204, 15; and where indicated, PMS, 0.4; K3Fe(CN)6, 18; or vitamin K3, 0.3 (added with 5 ;smoles sodium ascorbate). The reaction was run for 10 minutes. The reaction vessels were con- tinuously flushed with purified nitrogen gas during the illumination period. Methods.-Reaction mixtures were prevented from freezing by adding methanol to a final concentration of 10 per cent (v/v). The reactions were carried out in Warburg vessels, in an illuminated constant temperature bath filled with a mixture of methanol and water. Illumination was through a glass bottom, by a bank of 300 Watt Reflector Spot bulbs. Broken chloroplasts26 (PI8) were used in all experi- ments. The measurement of ATP and other experimental procedures have been described elsewhere.27 The mitochondrial fraction from leaves was prepared in the same manner as the "remaining particles" described in reference 27, except that the 1-min centrifugation step at 18,000 X g was omitted. Results.-Table 1 shows the formation of ATP by cyclic and noncyclic photo- phosphorylation at -80C. Three variants of cyclic photophosphorylation (equa- tion 1), catalyzed either by PMS14, or by FMN14, or by vit. K3, were compared with two variants of noncyclic photophosphorylation (equation 2), one with TPN and one with ferricyanide.25 At-8C, ATP formation was more than twice as high in the PMS system as in any of the other photophosphorylating systems. The experiments represented by Table 1 were carried out at a high light intensity (50,000 lux). A comparison made at different light intensities (Fig. 1) shows that the superiority of the PMS pathway at -8oC, over noncyclic photophosphorylation with ferricyanide and cyclic photophosphorylation catalyzed by vit. K3, increased with an increase in light intensity. At -8CC, the latter two pathways became light- Downloaded by guest on October 1, 2021 836 BIOCHEMISTRY: HALL AND ARNON Piaoc. N. A. S. I I 1 I I I I 7 - 4,000 Lux - - 4,000 Lux -/0 -0FMN -/ - - 5- K3 ~~~~~FeCy TPN N PMS -/0-5 0 5 /0/15 -/0-5 0 5 /0/15 degrees centigrade FIG. 3.-Cyclic and noncyclic photophosphorylation at a low light intensity as a function of temperature. The reaction was run for 14 minutes at an illumination of 4,000 lux. The-reaction mixture was the same as described for Figure 1 except that 1 mg chlorophyll was used and where indicated, 0.3 jsmole FMN (together with 5 ,Amoles ascorbate) and 9 ;&moles TPN were added. saturated at a lower light intensity, suggesting that in these two pathways, but not in the one catalyzed by PMS, the dark chemical reactions involved in ATP forma- tion became limited even at the low electron flux induced by low illumination. As shown in Figure 2, the formation of ATP in the PMS pathway was propor- tional to chlorophyll concentration at -80C, but at 15'C, ATP formation became saturated at a relatively low chlorophyll concentration. It seems likely that the apparent dependence on high chlorophyll at the low temperature reflects not a requirement for chlorophyll per se (it is probably already present in excess) but rather a dependence on an associated chloroplast component(s) which may partici- pate in the temperature-limited dark reaction.