Photosynthetic Phosphorylation Above and Below 00C by David 0

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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 photosynthetic 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 photosynthesis, 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 September 27, 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 possible 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 photosynthetic 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) containing 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 photophosphorylation in isolated chloroplasts were investigated over a range of temperature from - 100 to 15'C. The results of these experiments reported in this communication -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 September 27, 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 continuously 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 experiments. 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 photophosphorylation at -80C. Three variants of cyclic photophosphorylation (equation 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 September 27, 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 formation 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. The low molecular ratio (1/400) of cytochrome f to chlorophyll28 may be significant in this connection. Our proposed mechanism for photophosphorylation'5 envisages ATP formation as being coupled with electron transfer via the cytochrome components of chloroplasts. The differences, among the several systems, in the temperature-dependent steps that are involved in ATP formation are shown in Figure 3. Here the light intensity was kept low while the temperature was varied from -10° to 15'C. At this low light intensity, noncyclic photophosphorylation and the cyclic FMN and vit. K systems were superior to the cyclic PMS system at 15'C but inferior to it at - 10'C. Thus at 15'C- and at a low light intensity the dark reactions in the- vit. K and FMN systems were more effective than in the PMS system in converting radiant energy into the pyrophosphate bond energy of ATP. At a low light intensity ATP formation by the PMS pathway was independent of temperature in the range of -10° to 15'C. However, at a high light intensity ATP formation by the PMS pathway became temperature-dependent (Fig. 4). Thus, in the PMS pathway, the chemical reaction(s) of ATP formation seemed to keep pace with a low but not with a high electron flux induced by illumination. Table 2 gives a comparison between the effects of temperature on photophosphorylation and oxidative phosphorylation by a chloroplast fraction and by a mitochondrial fraction, respectively, both of which were isolated from the same leaves. A shift from 15" to -2°C has virtually stopped oxidative phosphorylation but had no effect on photophosphorvlation. Downloaded by guest on September 27, 2021 VOL. 48, 1962 BIOCHEMISTRY: HALL AND ARNON 837

/4 40,000 Lux

b0'L//V/2

LIGHT -/0 -5 0 5 /0 I5 FIG. 5.-Diagrammatic degrees centigrade representation of phosphorylation steps coupled FIG. 4.-Cyclic photophosphoryl- with electron flow in cvelic ation (PMS system) at a high light photophosphorylation. intensity as a function of tempera- Heavy black lines repre- ture. The reaction was run for 6 sent electron flow as cat- minutes at an illumination of 40,000 alyzed by FMN or vitamin lux. The reaction mixture was the K. Broken line represents same as described for Figure 1 ex- the "short cut" flow, cat- cept that 1 mg chlorophyll was used alyzed by PMS, which by- and ADP and K2HP3204 were each passes a phosphorylation increased to 20 Mmoles. site.

Concluding Remarks.-The "electron flow" mechanisms for cyclic and noncyclic photophosphorylation which we have proposed'5 include a combination of physical steps (temperature-insensitive) and chemical steps (temperature'sensitive). In cyclic photophosphorylation, the envisaged physical steps begin with photon capture by a chlorophyll molecule (bound to the chloroplast structure). The resultant excited state of chlorophyll is followed by a transfer of a "high-energy" electron from chlorophyll to a primary acceptor. The chemical steps are concerned with subsequent electron transfer to a cofactor and thence, via a chain of other catalysts, which include cytochromes, back to chlorophyll. One or more of these electron transfer steps is coupled with the formation of the pyrophosphate bonds of ATP. The same physical events were envisaged for noncyclic photophosphorylation but here the chemical steps included, in addition to ATP formation, the enzymatic reduction of pyridine nucleotide by the high energy electron "expelled" by excited chlorophyll. The electrons thus removed from the "cycle" are replaced by an external electron donor, which in the case of chloroplasts is normally water (OH-). Diagrammatic representations of these mechanisms are given elsewhere.23' 26 Evidence for temperature-independent, light-induced electron shifts in chlorophyll is found in the recent observations of Arnold and Clayton29 on chromatophores of photosynthetic bacteria, which showed reversible spectral changes in a temperature range from 300'K down to 1 K. Chance and Nishimura,30 working with whole cells of the photosynthetic bacterium Chromatium, observed a light-induced oxidation of cytochrome that was independent of temperature down to 77'K. These findings are in harmony with the view that photon capture during the primary photochemical act involves an electron transfer between cytochrome and chlorophyll. 15 Visualizing similar events in chloroplasts, it would appear that here also the Downloaded by guest on September 27, 2021 838 BIOCHEMISTRY: HALL AND ARNON PROC. N. A. S.

TABLE 2 EFFECT OF TEMPERATURE ON OXIDATIVE AND PHOTOSYNTHETIC PHOSPHORYLATIONS BY PARTICULATE FRACTIONS FROM LEAVES Oxygen ATP formed absorbed Centigrade (jsmoles) (Matoms) Oxidative phosphorylation, mitochondrial 150 2.5 3.4 fraction; gas phase, air -20 0.4 0 Cyclic photophosphorylation, chloroplast 150 3.2 - fraction; gas phase, nitrogen -20 3.6 In the oxidative phosphorylation system the reaction mixture included, in a final volume of 3 ml, a leaf mitochondrial fraction (see Methods), and the following in Amoles: Tris/acetate buffer, pH 7.4, 40; potassium-sodium-P'204, pH 7.2, 20; MgSO4, 10; ADP, 2; a-ketoglutarate, 45; plus 10 mg glucose and 75 units of hexokinase (Sigma, type III). The reaction was run for 90 minutes in the dark. 0.1 ml of 20% KOH was added to the center well of the Warburg vessels. In the photosynthetic phosphorylation system the reaction mixture included, in a final volume of 3 ml, chloroplast fragments containing 2.0 mg chlorophyll and the following in pmoles: Tris/acetate buffer, pH 8.0, 80; MgSO4, 10; ADP, 10; KHP3204, 10; and PMS, 0.1. The reaction was run for 20 minutes at an illumination of 4,000 lux; reaction vessels were continuously flushed with purified nitrogen.

light-induced, temperature-insensitive electron transfer (cf. charge transfer mecha- nisms3") involves a loss of an electron from chlorophyll and its replacement by an electron donated by cytochrome, which thereby becomes oxidized. The temperature-dependent steps would include, among others, the formation of ATP and the reduction of a "terminal"15 cytochrome, i.e., a cytochrome component that is adjacent to an excited chlorophyll molecule. These are steps which in our scheme'5' 23 are catalyzed, directly or indirectly, by FMN, vit. K or PMS. Nishi- mura,32 using the flashing light technique, has recently obtained evidence for dark, PMS-eatalyzed, chemical step(s) in cyclic photophosphorylation by bacterial chromatophor'es. The appreciable formation of ATP below 0C and the observed differences in temperature sensitivity may be useful in distinguishing between the component reactions of photosynthetic phosphorylation. The superiority of PMS as a cofactor of photophosphorylation at low temperature may be explained by the rapid inter- action of PMS with cytochromes,33 and the resultant bypass of one or more temperature-sensitive step(s) which are included in the cyclic pathway catalyzed by FMN or vit. K. We have suggested on the basis of other evidence23 that, as represented in Figure 5, the longer electron transfer route in the FMN or vit. K pathway has at least one more phosphorylation site. The data shown in Figure 3 (left) are consistent with this suggestion. The difference in temperature sensitivity between oxidative phosphorylation by leaf mitochondria and photosynthetic phosphorylation by chloroplasts (Table 2) provides further support for the concept that these are two distinct major sites for ATP formation in photosynthetic cells.34 It also gives rise to questions about photosynthetic phosphorylation as a possible source of cellular ATP at low temperatures, as for example, in the phytoplankton of polar regions35 or in conifers during cold winter seasons.

We are indebted to Miss Dorothy DeKiewiet for valuable assistance. * The experimental data in this article will form-part of a thesis to be submitted by this author in partial fulfillment of the requirements for the Ph.D. degree at the University of California at Berkeley. t Aided by grants from the United States Public Health Service and the Office of Naval Re- search. Downloaded by guest on September 27, 2021 VOL. 48, 1962 BIOCHEMISTRY: HALL AND ARNON 839

1 Calvin, M., Science, 135, 879 (1962). 2 Weissbach, A., B. L. Horecker, and J. Hurwitz, J. Biol. Chem., 218, 795 (1956). 3Jakoby, WT. B., D. 0. Brummond, and S. Ochoa, J. Biol. Chem., 218, 811 (1956). 4Racker, E., Arch. Bioch. Biophys., 69, 300 (1957). 6 Santer, M., and W. Vishniac, Biochim. Biophys. Acta, 18, 157 (1955). 6 Trudinger, P. A., Biochim. Biophys. Acta, 18, 581 (1955); Biochem. J., 64, 274 (1956). 7 Aubert, J. P., G. Milhaud, and J. Millet, Compt. rend., 242, 2059 (1956); Milhaud, G., J. P. Aubert, and J. Millet, Compt. rend., 243, 102 (1956). 8 Bergmanr F. H., J. C. Towne, and R. H. Burris, J. Biol. Chem., 230, 13 (1958). 9 McFadden, B. A., J. Bacteriol., 77, 339 (1959). 10 McFadden, B. A., and D. E. Atkinson, Arch. Biochem. Biophys., 66, 16 (1957). '1 Suzuki, I.; and C. H. Werkman, Iowa State College J. Sc., 32, 485 (1958); Arch. Biochem. Biophys., 77, 112 (1958). 12 Kornberg, H. L., J. F. Collins, and D. Bigley, Biochim. Biophys. Acta, 39, 9 (1960). 13 Malavolta, E., C. C. Delwiche, and W. D. Burge, Biochem. Biophys. Research Comms., 2, 445 (1960). 14 The following abbreviations are used: ATP, adenosine triphosphate; ADP, adenosine diphosphate; P, orthophosphate; PN, PNH2 oxidized and reduced forms of diphospho- or tri- phosphopyridine nucleotide (DPN or TPN); PMS, phenazine methosulfate (methyl phenazonium methosulfate); FMN, flavin mononucleotide (riboflavin phosphate). "I Arnon, D. I., Nature, 184, 10 (1959). 16 Arnon, D. I., M. B. Allen, and F. R. Whatley, Nature, 174, 394 (1954). 17 Arnon, D. I., F. R. Whatley, and M. B. Allen, J. Am. Chem. Soc., 76, 6324 (1954). 18 Arnon, D. I., F. R. Whatley, and M. B. Allen, Science, 127, 1026 (1958). 19 Frenkel, A. W., J. Am. Chem. Soc., 76, 5568 (1954). 20 Nozaki, M., K. Tagawa, and D. I. Arnon, these PROCEEDINGS, 47, 1334 (1961). 21 Thomas, J. B., and A. M. Haans, Biochim. Biophys. Acta, 19, 570 (1956). 22 Petrack, B., and F. Lipmann, in Light and Life, ed. W. D. McElroy and B. Glass (Baltimore: Johns Hopkins Press, 1961), p. 621. 23 Arnon, D. I., ibid., p. 489. 23a Jagendorf, A. T., in Survey of Biological Progress, ed. B. Glass (New York: Academic Press, 1962), vol. 4, p. 181. 24 Geller, D. M., and F. Lipmann, J. Biol. Chem., 235, 2478 (1960). 2 Arnon, D. I., M. Losada, F. R. Whatley, H. Y. Tsujimoto, D. 0. Hall, and A. A. Horton, these PROCEEDINGS, 47, 1314 (1961). "* Whatley, F. R., M. B. Allen, and D. I. Arnon, Biochim. Biophys. Acta, 32, 32 (1959). 27 Arnon, D. I., M. B. Allen, and F. R. Whatley, ibid., 20, 449 (1956). 28Davenport, H. E., and R. Hill, Proc. Roy. Soc. (London), 139B, 327 (1952). 29 Arnold, W., and R. K. Clayton, these PROCEEDINGS, 46, 769 (1960). 30 Chance, B., and M. Nishimura, these PROCEEDINGS, 46, 19 (1960). 31 Szent-Gyorgyi, A., Introduction to Submolecular Biology (New York: Academic Press, 1960), p. 54. 32 Nishimura, M., Federation Proc., 20, 374 (1961). 33 Massey, B., Biochim. Biophys. Acta, 34, 255 (1959). 34 Arnon, D. I., Science, 122, 9 (1955). 35Anonymous, IGY Bull. No. 54, 12 (1961). Downloaded by guest on September 27, 2021