Phowsynthesis Research 48: 19-23, 1996. (~) 1996 KluwerAcademic Publishers. Printedin the Netherlands. Reflection
Bill Arnold's concept of solid state photosynthesis and his discoveries David Mauzerall The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 6 December 1995; accepted in revised form 25 January 1996 Key words: Arnold, cyrogenics, delayed luminescence, electroluminescence, photosynthetic unit, solid state model, thermoluminescence, turnover time
Abstract Bill Arnold's concept of photosynthesis as a solid state, as opposed to solution, phenomenon led him to an amazing series of fundamental discoveries.
Introduction The conceptual framework upon which we fit observations predetermines in many ways both our interpretations of data and the crucial novel next step or discovery, the engine of science. Excellent examples of this process can be seen in the discoveries of Bill Arnold in photosynthesis. When Arnold began his work in about 1930, the basic facts of photosynthesis had been established. The concept of sequential light and dark reactions was accepted. The biochemical metabolic approach predominated: photosynthesis was a special case of anabolic metabolism. If one could identify the 'chlorophyll enzyme' the problem would be solved. A completely different view was to be adopted by a few physically oriented workers: photosynthesis occurred in a complex unit and the mechanism was closer to that being developed in solid state physics to explain photoconduction. In Bill Arnold's mind and hands, this latter concept led to a remarkable series of discoveries.
Turnover time and the photosynthetic unit Arnold began his career with the great experimentalist Emerson. They re-oriented the problem of the light and dark reactions of photosynthesis by asking specifically what was the turnover time of photosynthesis (Emerson and Arnold 1932a). Based on knowledge
of photochemistry, the light reaction was expected to be fast and to have a small activation energy, while the dark reaction would be slow and be much more dependent on temperature. Warburg (1919) had earlier observed that intermittent light produced more photosynthesis than an equal amount of continuous light at high intensities, an effect first observed by Brown and Escombe (1905). A kinetic interpretation of this effect is that some light is 'wasted' during the time of the dark reaction. A key contribution by Arnold was to develop a neon flash lamp that gave a much shorter flash, ,,~10/zs, than the previously used rotating sectors (,,~3 ms) and that could be fired 30 times per second. Jack Myers has written an exquisite precis of 'THE experiment' (Myers 1994). I omit technical details since he has presented them so well. The remarkable lamp and power supply that Arnold assembled could not be fired fast enough to determine the turnover time of oxygen production at room temperature. But by cooling to 1 °C, a turnover time of 30 ms was measured, with the same oxygen yield per flash as at room temperature. This unique experiment set the time scale for the temperature sensitive dark or Blackman reaction and indicated the temperature insensitivity of the light reaction. Since chlorophyll was the photosensitizer for these reactions, Emerson and Arnold asked how many chlorophyll molecules were present for the reduction of one molecule of carbon dioxide (Emerson and Arnold 1932b). Although they measured oxygen, photosyn-
20 thesis in those days meant reduction of carbon dioxide and the terms were used interchangeably. We now know this is not a justifiable assumption. The key concept required for the proposed measurement was that of the single turnover flash: a flash of duration far less than the turnover time, yet bright enough that all the units would be excited. And it had to be fired 30 or so times per second. It is a measure of Arnold's abilities that these experiments were not properly repeated until two decades later. As Myers stresses, the experiment additionally required the quantitative measure of the chlorophyll content of cells with a visual photometer using a neon emission line, also a distinct first. Emerson and Arnold found that the ratio of chlorophyll present to oxygen molecules formed was about 2500, suggestive of a large photosynthetic unit (PSU). Since the received wisdom of the day was that there was one chlorophyll per enzyme, the obvious explanation was that this was the result to be expected when physicists toyed with biology: 99.9% of their Chlorella were dead. Somewhat more seriously, there were the problems of ensuring light saturation, of errors in the oxygen or chlorophyll determinations, etc. Arnold and Kohn (1934) addressed several of these problems directly, but resistance remained. These objections were reinforced when other workers, not appreciating the crucial concept of single turnover flashes, repeated the experiments with multi ms flashes and obtained smaller ratios (Tamiya and Chiba 1949). Myers (1994) presents evidence that the star-crossed theoretician of photosynthesis, James Franck, had an 'implacable opposition' to the concept of a PSU. The PSU was a concept far ahead of its time. Yet, the first successful theoretician of photosynthesis, Hans Gaffron, had already proposed with Wohl (Gaffron and Wohl 1936) that hundreds of chlorphylls were assembled as a unit to carry out photosynthesis. He based his argument on the short time required for oxygen production to reach a steady state in dim light, a time too short for all chlorophylls to have been excited. We now know this is a tricky argument since in very dim light no oxygen can be made because of decay of the individual S state comprising the oxygen forming cycle. The limited sensitivity of the Warburg apparatus prevented observation of this potentially confusing fact. The crucial aspect of energy transfer between the molecules comprising a PSU was added later by Arnold (Arnold and Oppenheimer 1950), and proven by Duysens (1951) some time later. The simple concepts of turnover time and size of a PSU have been much embellished since that time. Pho-
tosynthesis turns out to be a somewhat non-linear process - just non-linear enough to have kept researchers busy for some six decades since 'THE experiment' (Myers 1994). The 'fastest' or transient turnover time, best measured by single turnover pump-probe experiments, is ,,~200 #s and is now known to be the regeneration time of the primary quinone acceptor (Forbush and Kok 1968; Mauzeral11972). This time is essentially a constant of photosynthesis, being almost the same for a wide range of plants, algae and cyanobacteria (see e.g. Mishkind and Mauzerall 1980). The 'slow' or steady state turnover time(s) of photosynthesis is ,,~ 10 ms and is much more variable. It is measured by the Emerson and Arnold repetitive single turnover flash method or by estimates from steady state rates together with photon absorption rates and quantum yields. The difference between these turnover times is caused by differing numbers of intermediates in the sequence of reactions and in particular by the size and redox level of the quinone pool linking the two photosystems. The fact that 'the' turnover time of photosynthesis is a variable, dependent on past illumination among other things, has caused much confusion. This richness uncovered by work on the turnover time is just one legacy of 'THE experiment'. The concept of the size of the PSU has also undergone many refinements. The simplicity of the concept was retained, following the hypothesis of two photosystems by Hill and Bendall (1960), by assuming that a unit contained both photosystems to achieve maximum efficiency (Myers and Graham 1963; Clayton 1963). Evidence that the photosystems occurred in differing portions of the thylakoids (Anderson 1975; Staehelin et al. 1976) and the finding of non-unity ratios of the two photosystems (Melis and Brown 1980; Falkowski et al. 1981) forced a re-assessment of the basic concept. Moreover, the idea that the chlorophyll antennae could serve more than one unit, or reaction center, i.e. that the excitation energy could ignore a closed trap and go on to another, espoused by Joliot and Joliot (1964), further weakened the concept of a monolithic PSU. To add to the confusion it became customary to divide the Emerson Arnold unit by various numbers to obtain the sub-unit size: 2 for 2 photosystems, 4 for 4 electrons to make oxygen, 2 x 4 because of two photosystems, etc. A tower of babel had arisen. The solution to this problem was understood by Arnold, but was difficult to accomplish for a technical reason: the lack of a monochromatic single turnover flash of sufficient energy (photon flux). Excitation with flashes of varying energy causes a Poisson distribution of excitation in the
21 collection of PSUs. The average excitation ('hits') is given by the product of the photon flux and the optical cross section of the PSU. On increasing the flash energy to saturation of oxygen yield and fitting the data to the cumulative one hit Poisson distribution, the target size or optical cross section can be determined (Ley and Mauzerall 1982). A preliminary measurement of this kind, published by Kohn (1936), gave a rather large size for the unit. Pulsed lasers finally allowed the measurement to be carried out. The result was a cross section of 250 .~2 at 590 nm, several hundred times larger than that of a single molecule. The usefulness of such a number is its unambiguous identification of all molecules and only those molecules contributing to the observed photosynthetic reaction, for example oxygen formation. Monochromatic light is required so that the optical cross section(s) of molecule(s) at that wavelength can be determined in vivo. The ratio of these cross sections then results in the unambiguous 'size' of the oxygen unit: 250 Chl to form 02 in Chlorella. The measurement is absolute in that it is independent of other absorbing molecules, including dead units, as long as the sample is optically thin. The ratio of the Emerson Arnold PSU, 2500 to the oxygen unit size, 250, is the quantum requirement of photosynthesis, 10. The quantum requirement so derived is the same as that found by Emerson and Lewis (1943 ) some time ago. Thus the magic number by which to divide the Emerson-Arnold PSU to obtain a reaction PSU is the quantum requirement of that reaction. The saturation methods can be applied to other photosystems, and a complete inventory of chlorophyll in the photosystems of Chlorella has been obtained (Greenbaum and Mauzerall 1991). The concept of the photosynthetic unit and its associated antennae pigments implied a reactive trap or reaction center where the excitation energy was converted to chemical energy. These centers were first identified optically by Duysens (1989) and by Kok (1956). They were then reified by Reed and Clayton (1968). The rest is modern history.
Delayed luminescence and thermoluminescence Following illumination some materials emit light on a far longer time scale than the ns time scale of fluorescence. The emission is called delayed luminescence if it is at the same wavelengths as fluorescence and phosphorescence if it is at longer wavelengths. When illuminated at low temperatures and slowly warmed
the former materials emit increased light at characteristic temperatures, yielding a 'glow curve' of thermoluminescence. In contrast the phosphorescent lifetime is temperature independent when diffusive quenching events are eliminated. Arnold's discovery of these two powerful tools for the study of photosynthesis, delayed luminescence and thermoluminescence, involved the productive mixture of serendipity and the prepared mind. In attempts with Strehler to measure ATP production in chloroplasts by the ATP-luciferin luminescence (Strehler and Arnold 1951 a,b; Strehler, this issue), a signal in the control was traced to the chloroplasts alone following illumination. Arnold and Strehler interpreted this delayed luminescence as recombination luminescence. This was by analogy with photoinduced separation of charges in solid state material and the subsequent recombination of the charges with emission of a photon. His knowledge of the thermal glow curves of these substances led him to try the same measurements on chloroplasts (Arnold and Sherwood 1957). The result was the striking thermoluminescence curve, which spells out the energy levels of 'traps' in the system (Arnold and Azzi 1968). These data were interpreted on a suggestion by DeVault and Govindjee as the thermally activated reversal of photosynthetic reaction steps (DeVault et al. 1983). The study of the intricacies of these glow curves have contributed much to our understanding of photosynthetic reactions and that work is discussed by Vass and Govindjee in this issue.
Electroluminescence Arnold again claims that serendipity was responsible for his discovery of electroluminescence with Azzi (Arnold and Azzi 1971). Again, however, the experiment was designed with the solid state in mind: a current through the chloroplast might remove a component of the glow curve. In fact, it was observed that applying voltage across a suspension of chloroplast particles produced a burst of luminescence. It was then found that it was the voltage across waterswollen blebs of chloroplast membranes that gave the large light enhancement. This was not an experiment that would have occurred to the myriad spectroscopists who were busily, and successfully, attacking steps in the photosynthetic process. The study of electroluminescence has not only added to our understanding of charge separation in the photosystems but has made us appreciate the large electrostatic effects in the hetero-
22 geneous dielectric systems that make up the PSU. This work is reviewed by van Gorkom in this issue.
Cryogenic photosynthesis The radically different concept of photosynthesis as a solid state process was required to allow one to conceive of the possibility that a photosynthetic reaction could occur at the temperature of liquid helium. This is just what Clayton and Arnold set out to measure in 1960 (Arnold and Clayton 1960; Clayton and Arnold 1960). They were able to do the experiment because of the development by Duysens and others of spectroscopic methods of detecting early photochemical steps in photosynthesis. The optical absorption change of bacteriochlorophyll on illuminating a sample of photosynthetic bacteria was observed at 1 K. A more striking justification of a solid state view of photosynthesis is hard to imagine. It was the culmination of the original Emerson and Arnold experiment where the dark chemical or Blackman reaction was made observable by cooling, slowing it but not affecting the photochemical reaction. It was also a surprise to many scientists that a photochemical electron transfer, and a biological one to boot, could proceed at this low temperature. This observation convinced many that Arnold's view of solid state photosynthesis was relevant, not idiosyncratic. It also made one think that quantum mechanical tunneling might be involved in the primary reaction of photosynthesis. In the simplest case, the tunneling process is completely independent of temperature. Devault and Chance (1966) showed that the rate of reduction of the photooxidized pigment by a cytochrome became temperature independent below 100 K, and McElroy et al. (1974) showed that the reverse electron transfer between the stabilized acceptor, a quinone anion, and the photooxidized pigment cation was completely temperature independent from 77 to 1.4 K. These studies, and the determination of the atomic structure of a bacterial reaction by Deisenhofer et al. (1984) established that the mechanism of the primary electron transfer photoreaction in photosynthesis was that of quantum mechanical tunneling. The X-ray structure showed that the donors and acceptors were far from van der Waal's contact, a crucial fact already established by EPR studies of the reaction center. Thus, Arnold's view of solid state photosynthesis was substantiated.
Arnold's concept of solid state photosynthesis Like most original ideas, such as van Niel's concept of variable donor photosynthesis (van Niel 1935), Arnold's concept of solid state photosynthesis (Arnold 1965) turned out to be partially right and partially wrong. It is certainly far more correct than the view prevalent at that time: the 'solution' view that photosynthesis occurred at chlorophyll on a 'photoenzyme' which reduced carbon dioxide and oxidized water. Arnold's prediction that the separation of charge is the crucial primary process was perfectly correct. The present summary of Arnold's contributions and their effect on the direction of research and on the eventual solution of basic problems in photosynthesis clearly shows the value and importance of his concept. Where it failed was in the direct analogy with photoeffects in the solid state, such as in semiconductors. The charge separation occurs not in a lattice of pigment molecules, with consequent migration of charge in conduction bands, but between highly specific molecules carefully positioned by protein structure in the reaction center. The surrounding 'lattice' of pigments is used as an antenna to increase the capture cross section for photons. A physical reason for this difference between Arnold's concept and reality is that the wavefunctions of organic crystals are highly localized, i.e. they are dielectrics, not semiconductors. Although organic semicondutors and conductors are now known, they require highly specialized structures and partially occupied molecular orbitals. A biological reason for this difference is that the evolution of photosynthesis most likely occurred via molecular solution photochemistry and progressed through aggregates in lipid bilayers to the highly organized systems now present (Mauzerall 1973). In any case this error fades in significance when compared to the far reaching positive contributions of Arnold's concept of solid state photosynthesis.
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