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PHOTOSYNTHESIS AND ENERGY TRANSFER BY E. RABINOWITCH Photosynthesis Laboratory, Botany Department, University of Illinois, Urbana, Ill. Received March 14, lQ.57
Resonance energy transfer of two types can occur in photosynthesizing cells: (a) repeated, “homogeneous” transfer from chlorophyll molecule to chlorophyll molecule, which could cover several hundred (or thousand) molecules before the energy is re-emitted or dissipated; this migration may be useful in permitting photons, absorbed by a large number of chlorophyll molecules, to initiate enzymatic reaction chains in a much smaller number of enzyme sites; (b) one-time, “heterogeneous” excitation energy transfer from accessory pigments (carotenoids, phycobilins) to chlorophyll, which may permit certain cells to utilize for photosynthesis light only oorly absorbed by chlorophyll itself. This paper reviews observations on the packing of pigments in chloroplasts, and on t i e life time and yield of chlorophyll fluorescence in vivo-data needed to estimate the probable range of “homogeneous” energy migration. I n the last section, a brief summary is given of the observations of sensitized chlorophyll fluorescence in vivo, which indicates the efficiency of “heterogeneous” energy transfer in the living cell.
I. Introduction Photosynthesis is light-induced reversal of cellular respiration. It has two main aspects-organic synthesis (reduction and condensation of carbon dioxide to carbohydrate), and energy storage (conversion of light energy into potential chemical energy). At present, there are reasons to believe that these two phenomena, although closely tied together, are separable. First, the photochemical mechanism lifts hydrogen from its low reducing potential in water t o a potential high enough to reduce C-0 bonds; then, the organic-synthetic mechanism utilizes this hydrogen (perhaps, together with some high-energy phosphates, obtained by partial reversal of the photochemical process) for the synthesis of carbohydrates. Even if-as Franck believesl-the photochemical system transfers hydrogen atoms directly to an intermediate of the carbohydrate synthesis (such as phosphoglyceric acid), and not to an intermediate hydrogen-carrier, such as TPN-as the majority of other workers in the field believe-it remains true that the connection between the photochemical and the synthetic process can be uncoupled, and the photochemical system made t.0 unload light-activated hydrogen on a substitute acceptor (so-called Hill reaction). The organic-synthetic system, which produces carbohydrates from carbon dioxide, in its turn, appears to be able to operate with reductants other than the light-produced one. This conclusion is suggested by the metabolism of chemosynthetic bacteria and Gaffron’s observation that certain normally photosynthetic green algae acquire, after anaerobic incubation, a capacity for ‘(chemosynthetic” reduction of carbon dioxide. I n its net result the organic phase of photosynthesis is equivalent t o the reversal of the glycolytic stage of respiration, including the Krebs cycle; however, C(14)-studies have shown that this reversal is not precise, but goes through a different sequence of intermediates. The energy-storing phase of photosynthesis amounts, in its net chemical result, to a reversal of the last stage of respirationthe ‘(downhill” transport of hydrogen, from a reduction potential of about +0.35 volt in pyridine nucleotides, to a reduction potential of about -0.81 volt in water. (1) J. Franck, in “Research in Photosynthesis,” (Proceedings of the Second Gatlinburg Conference on Photosynthesis of the N.R.C., Oct. 1955, in press, Interscience Publishers, Inc., New York, N. Y.).
I n this case, we have even less reason than in the organic synthesis to anticipate that the endothermal process will be the exact reversal of the exothermal reaction (which goes via riboflavin, the cytochromes, etc.), since the latter proceeds in ((smallsteps” (AF 0.3 e.v.) to assure effective metabolic utilization of liberated energy via ATP-formation; while the former must utilize much larger energy unitsquanta of visible light (2-3 e.v.). This part of photosynthesis, immediately associated with the primary photochemical process, is, a t present, the least understood, and-for a physical chemist-also the most interesting one. I n the non-photochemical stage of photosynthesis, the products of the photochemical process must undergo a sequence of enzymatic transformations. The enzymes are large protein molecules, and several dozen different ones are needed for complete cell metabolism. Therefore, each enzyme can be represented in the cell only by a relatively small number of molecules-with a concentration of the order of mole/l. or less. The “working times” of enzymes are short enough for these relatively small amounts to assure the necessary speed of all metabolic processes. Of these, photosynthesis is by far the fastest one-up t o 20 or 30 times faster than cellular respiration-corresponding to a substrate turnover of the order of m.1. sec. - l , The maximum rate a t which the primary photochemical process can run is, on the other hand, limited by the light-absorbing capacity of the pigments. The molar absorption coefficients of even the most intensely colored organic pigment do not lo8 cm.2 m.-I. T o achieve good abexceed a sorption of incident light (say, log lo/l11 0.3) in a single cell, whose thickness is of the order of 10-4 cm., the concentration of the pigment in this cell must be c O.3/otd 0.03 m. Chlorophyll is in fact present, in green cells, in concentrations of this order of magnitude. We thus see that photosynthesis involves absorption of photons by a very large number of pigment molecules, and transformation of the primary photochemical products by a much smaller number of enzyme molecules, perhaps one per several thousand pigment molecules. Efficiency measurements indicate that under the most favorable conditions, no significant fraction of the absorbed energy quanta is entirely lost for photosynthesis-although, in all probability, not more than 25-30% of the energy of each utilized quan-
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c.
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existence in the photosynthetic system of a catalytic component whose normal concentration is about 42000th of that of chlorophyll, n being a small whole number indicating how many molecules of the substrate must be transformed by the yieldlimiting enzyme to permit the liberation of one molecule of oxygen (n = 4 is a plausible value, because four hydrogen atoms have to be transferred from HzO to C 0 2 to produce one molecule 0 2 ) . I n the picture presented in the preceding section, the number N (“ 2000) could be simply the ratio of the concentrations of chlorophyll and of the enzyme which transforms the primary photochemical product. To account for the fact that prolongation of intervals between flashes does not increase the yield of brief flashes above the above-mentioned P*maxvalue, we would have to assume that this primary product (or products) are unstable, and disappear by back reactions (or recombinations) unless they are taken care of immediately by the enzyme (“immediately” meaning “within a time short in comparison to the operating time of the enzyme,” which other experiments show to be of the order of sec.). If this were the case, the “limiting” enzyme could operate only once in the wake of each flash; by the time it has completed its work on the first batch of substrate, the rest of the latter would have disappeared by back reactions. Gaffron and Wohl suggested, however, that the limiting enzyme, instead of receiving its substrate by migration of chemical intermediates from the 2000/n chlorophyll sites, is activated by energy migration within a “photosynthetic unit” of 2000/n chlorophyll molecules. Migrating energy quanta which find the enzymatic reaction site associated with the unit “busy,” are de-activated (by conversion of electronic excitation energy to vibrations) , before the site becomes available again. The “unit” can be treated either as a particle (or an “island” in a two-dimensional layer), or as a statistical magnitude, indicating the average reach of energy migration within a larger (three- or twodimensional) array of pigment molecules; in the latter interpretation, an excitation quantum that finds the nearest reaction site occupied, must have a very low probability of reaching the nearest vacant site before being dissipated. The hypothesis of the photosynthetic unitwhether the latter is taken to mean a true particle, 11. Flashing Light Experiments or a statistically favored region-is a suggestive The hypothesis of resonance energy migration in one, but so far it could not be supported (nor could photosynthesis was first proposed in 1936 by Gaf- it be disproved) by direct experimental evidence. fron and Woh13 to explain the results of Emerson It is difficult to think of experiments which would and Arnold’s experiments4 on the kinetics of photo- provide an unambiguous confirmation (or clear synthesis in flashing light. The main result of refutation) of the existence of such a unit. We can, studies of this type is that the maximum amount of however, estimate the probability of the postulated photosynthesis, P*max, which can be achieved (in energy migration from the study of the density “normal” plants) by a single, very brief flash of light and spatial distribution of pigment molecules in the (tf < sec.) is P*,a, = ChZo/N, where Chlois the living cell, combined with theoretical and experitotal number of chlorophyll molecules present and mental evidence concerning the occurrence and N 2 X lo3. ( P * m a x is measured in molecules of O2 extent of energy migration in appropriate model liberated per flash.) This relation suggests the systems. (2) 0. Warburg, et al., Z. Nalurforschung, 6b, 285, 417 (1951); 111. Structure and Density of the Pigment System Naturwiss., 89, 185 (1952). Numerous investigations have been carried out (3) H.Gaffron and K. Wohl, ibid., 24, 81, 103 (1936). in the last ten years dealing with the submicro(4) R. Emerson and W. Arnold, J . Gen. Physiol., 15, 391; 16, 191 (1933). scopic structure of chloroplasts, which are the tum can be recovered as chemical energy at the end of the reaction sequence. [Warburg’s contention2 that this conversion can be 100% effective, i.e., that in photosynthesis we encounter a fast-working, complex chemical machinery, which operates without any friction losses whatsoever, is hard to believe for a physical chemist!] In “ordinary” photochemistry, the absorption of a photon produces a chemical change, either in the absorbing molecule itself, or in a molecule with which the absorber gets into contact during the excitation period. If the mechanism of photosynthesis were similar, each light-excited chlorophyll molecule would produce, in its site (since the chlorophyll molecules are probably bound to a protein surface), one molecule of the primary photochemical product (or two molecules of two primary products, e.g., an oxidation and a reduction product). The products formed in several hundred (or several thousand) sites would then have to diffuse to the one nearest enzyme molecule (since the latter, too, probably is bound t o a certain cell site); under favorable conditions (when the over-all process is not too fast), this diffusion must be accomplished practically without losses (which could be caused by back reactions of one, or re-combination of two, primary products). There is nothing impossible in this picture; but an alternative has been suggested which appears tempting to a physicist or physical chemist : namely, that instead of the intermediate products, the quanta of energy themselves migrate, by socalled resonance transfer, through the pigment system, until they reach an enzymatic reaction site. This purely physical mechanism of energy transport seems to be less cumbersome-and potentially more efficient-than the more trivial mechanism of migration of energy-rich photochemical intermediates. In favor of the hypothesis of energy migration as the mechanism by which a larger number of pigment molecules is coupled to a much smaller number of enzyme molecules, one can quote the fact that such migration is inevitable whenever pigment molecules are packed as densely as they are in the chromophores of photosynthesizing cells; and the cells may as well utilize this phenomenon to good purpose.
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Fig. 1.-A
single granular chloroplast of maize, fixed and sectioned, showing relation of grana to lamellae (after Vatter).
chlorophyll-bearing cell structures. Electron-microscopic observations made on disintegrated cell material have suggested, for a while, that the most important structural element in the chloroplasts of the higher plants are grana-cylindrical bodies about 0.5 .U in diameter and perhaps 0.3 p in height; these grana were often seen to disintegrate further into piles of discs, each about 0.01 p thick, suggesting that grana are built like club sandwiches, probably of alternating proteinaceous and lipoidic layer~.~ (5)
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Cf. E. I. Rabinowitch, ”Photosynthesis and Related Processes,”
Grana (or, at least, single discs) could be observed in electron-micrographs of chloroplast material from a large number of species; however, some photosynthesizing cells seemed to contain no such bodies; this was true particularly of many species of algae. More recently, the technique of fixing cells with osmic acid and slicing them very thinly for electron-microscopic observation has been perfected; the study of chloroplast preparations treated in this Vol. 11, 2, Chapter 37A, Interscience Publishers, Ino.. New York, N. Y., 1956.
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wag has shown the role of grana in a new light, The universal structural element shown by such chloroplast sections are lamellae (each about 0.01 p thick), which run through the whole chloroplast. Depending on the species and the physiological state of the cell, these lamellae may form more or less pronounced agglomerations of approximately cylindrical shape-regions where the lamellae are reinforced and often split in pairs. These reinforced cylindrical sections of the lamellar system appear as “grana” in the micrograms of disintegrated cells. Figure 1 shows, as an example, a section of a chloroplast of corn (Zea mays) according to Vatter.6 I n higher plants, granular structure is typical of mature, photosynthetically fully active cells; in algae-where chloroplasts do not usually have the regular ellipsoidal shape characteristic of the higher plants-the lamellar structure alone is generally recognizable. The .single chloroplast of Chlorella, the green alga on which a large part of the quantitative studies of photosynthesis have been carried out, has the shape of a bell, and the lamellae follow its Outline.’ The composition of the chloroplasts (about 2/3 Droteinaceous and lipoidic material), and various histological experiments, suggest that ’the lamellar system consists of alternate layers of more (or exclusively) proteinaceous, and of more (or exclusively) lipoidic composition. The osmium is precipitated preferentially on lipoids, so that the heavier layers in Os04-fixed preparations are those containing more lipoidic material, According to this (and other) evidence the grana must be richer in lipoids than other parts of the chloroplasts. The chlorophyll also accumulates in the grana, as indicated by observations in the fluorescence microscope, and by electron-microscopic proofs of preferential precipitation of silver on the grana in the socalled Molisch reaction (chlorophyll-sensitized reduction of AgNOa by ascorbic acid in light). The chlorophyll molecule has the shape of a kite with a square flat “head” and a flexible “tail.” The head is an aromatic, tetrapyrrole ring system (porphin). It contains a Mg atom in the center, and a carbonyl group in a short side chain, and therefore possesses a certain polarity; the tail is a long, almost saturated, entirely lipophilic “phytol” chain (Fig. 1A). It has been suggested-with considerable plausibility-that chlorophyll molecules will tend to arrange themselves on protein-lipoid interfaces, with porphin heads attached to the proteinaceous, and phytol chains dipping into the lipoidic layer. From electron micrographs, one can estimate roughly the total area of the protein-lipoid interface in a chloroplast. One can then compare this area with the number of chlorophyll molecules present in the chloroplasts and calculate the surface area available for each chlorophyll molecule. The calculations are very rough, because one has to use chlorophyll determinations made on one species, chloroplast and grana counts made on another one, and electron micrographs prepared from a third
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(6) A. E. Vatter, Thesis, Univ. of Illinois, 1955; cf. E. I. Rabinowitch, ref. 5, Chap. 38. (7) P. A. Albertson and H. Leyon, E z p t l . C.42.Res., 7,288 (1954). (8) J. B. Thomas. 0. H. Blaauw and L. N. M. Duysens, Biochim. Biophys. Acta, 10,230 (1953).
CHI’
\ CH2
Fig. 1A.-Structure
/CH-cHa
\
CH,
/””
CHs
;CH-CHa
\
of chlorophyll-a molecule.
one. [An exception is Euglena, recently studied by Wolken, Palade and co-workers9; but Euglena is a part-plant, part-animal, which even in its photosynthetic form shows a much smaller chlorophyll content than typical plant cells.lo] Preliminary estimates suggest that the interface area available for chlorophyll in the chloroplasts of typical plant cells is of the order of 1 mp2 per molecule. lo Crystalline monolayers of chlorophyll on water require an area of about 0.5 mu2per molecule: while compressed amorphous monolayers cover 1.OG mv2 per molecule (Jacobs, et al.“). (9) J. J. Wolken and G . E. Palade, Nature, 170, 114 (1952); Ann. N . Y . h a d . Sci., 56, 873 (1953); J . Gen. Physiol., 37, 111 (1954). (10) Quite recently, chlorophyll assays and surface estimations have been made by Thomas, Minnaert and Elbers (Acta Botanica NeerZandica, 5 , 315 (1956)) for chloroplasts of four plant species, and for one species of bacteria: the results varied between 0.8 and 3.8 mc’ per chlorophyll molecule (while the chlorophyll amounts and the arena available in a chloroplast, varied by as much as a factor of 104). (11) E. E. Jacobs, Thesis, Univ. of Illlinoie. 1952; Amh. Biochem. Biophys., in press; E. A. Hanson, PTOC.Roy. Acad. Amsterdam, 40,
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IV. The Lifetime of Excitation There are two ways to estimate the lifetime, 7 , of excited pigment molecules. One is to measure directly the decay of fluorescence; the other is to calculate the “natural” life time of excitation, r, by integration of the absorption curve, to measure the fluorescence quantum yield, p, and to calculate T from the well-known equation
0.14
0.12
;0.10
k
Zo.oe W D
7
0.06 0.04
0.02 0.00 WAVELENGTH IN rnp.
Fig. 2.-Absorptibn spectra of monolayers of chlorophylla (colloidal, curve 2;. crystalline-the precise nature of this layer is uncertain; it may be not uniformly monomolecular or bimolecular-, curve 3) and of ethyl chlorophyllide (crystalline, curve 1); curve 4 is the absorption spectrum of the same pigments in acetone. The ordinates of curves 1-3 are optical densities of single monolayers (after Jacobs, Vatter and Rabinowitch).
=
(1)
Po70
The direct measurement of 7 has been carried out in our laboratory by Brody,13 using a straightforward oscilloscopic method, with a small hydrogen lamp producing light flashes lasting 700 mp). The transfer competes effectively (but not 1 0 0 ~ oeffectively!) with the emission of fluorescence by the red and blue pigments, whose quantum yield is reduced, in consequence of this transfer, from 6080% in solution, to -10% in vivo. BrodyI3 has determined the time curves of the excitation of chlorophyll-a fluorescence by light absorbed by this pigment, and by light absorbed predominantly by phycoerythrin and transferred to chlorophyll a-presumably, via phycocyanin as intermediate. He found (cf. Fig. 4) that the onset of sensitized fluorescence is delayed, compared to that of directly excited fluorescence, by about 0.15 mp sec. This observed value agrees with the transfer time one can calculate from the reduction of the fluorescence yield of the phycobilins in vivo, and their excitation time in vitro. Much of the experimental work described in this article was carried out a t the Photosynthesis (22) H. J. Dutton, W. M. Manning and E. M. Dugper. THIS JOURNAL.47, 308 (1943). (23) L. N. M. Duysens, Thesis, Univ. Utrecht, 1962. (24) C. 8. French and V. K. Young, J . Gen. Physiol., 35, 873 (1952); also in “Biological Edects of Radiation,” Vol. 3, McGraw-Hill Book Co., Inc., New York, N. Y.. 1956.
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Laboratory of the University of Illinois by E. E. Jacobs, A. S. Holt, P. Latimer and S. S. Brody, and by the electron microscopist a t the University of
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Illinois, A. E. Vatter. The assistance of the Office of Naval Research in this research is gratefully acknowledged.
