Photosynthesis Research 21: 61-79, 1989. © Kluwer Academic Publishers. Printed in the Netherlands.

Personal Perspectives

The discovery of the two photosynthetic systems: a personal account* L.N.M. DuYSENS Department of Biophysics, Huygens Laboratory of the State University, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 6 December 1988

Key words: absorption difference spectrophotometry, cytochromes, history, photosystems, quantum requirement, reaction centers Abstract

A personal account is given of research leading to the discovery of the two photosystems in oxygenic photosynthesis. The following topics are discussed: transfer of excitation energy to (bacterio)chlorophyll and evidence for two pigment systems, the introduction of absorption difference spectroscopy, the discovery of reaction centers, cytochromes and other intermediates as photosynthetic electron or hydrogen transfer agents. Also discussed are chromatic transients and enhancement effects, phenomena indicating the existence of several photochemical but not necessarily major photosynthetic reactions. Various hypotheses or speculations by Eugene Rabinowitch, Robert Emerson, and Robin Hill are critically discussed. The discovery is described of the opposing effects of excitation at two wavelengths on the redox reactions of cytochrome f and other redox components, giving evidence for the existence of an electron transfer chain containing two major photochemical reactions in series, photoreactions 1 and 2, driven by two pigment systems. Finally it is argued that, after the successful introduction of absorption difference spectroscopy, the discovery of the two photosystems would probably have occurred within a decade, even in the absence of previous suggestive experiments or speculations.

1. Introduction

Govindjee kindly invited me to write a historical, possibly autobiographical review, on any subject in photosynthetic research. I considered to treat in some depth the early research on one of the main three subjects I worked on: 1) paths and mechanism of electronic excitation transfer, 2) primary and associated reactions and 3) the two photochemical systems in oxygenic photosynthesis. I chose the last subject because its discovery was partly based on insights gained by studying the first two subjects and shows the most interesting interaction between different scientists using different * This manuscript was completed on November 29, 1988 t Correspondence: Ch de Bourbonlaan 2, 2341 VD Oegstgeest, the Netherlands

experimental approaches and ways of thinking. I tried to recapture the reasonings and points of view I had at that time. The other two subjects are discussed in so far as needed as background. Only those biographical aspects will be mentioned that presumably significantly influenced my later scientific work.

1.1. Preparatory years Until my eighteenth year, I grew up and was educated in a Roman Catholic environment in the south of the Netherlands. I had, however, not accepted the prevailing specific religious and political beliefs and many accompanying convictions, because these did not appear to have a rational basis. Furthermore, I did not blindly follow people i n

Photograph of Dr E.I. Rabinowitch (in bow-tie) and the author taken at a lunch at the first Gatlinburg conference on photosynthesis, October 1952.

64 authority, because I often had seen them making errors of judgment or reasoning. I was deeply interested in how nature, including people, functioned, and tried to obtain insight by critical imagination, that is imagining various kinds of reasonably self-consistent models and selecting, if possible, one which fitted best with experience and with reasonably established knowledge, such as that obtained in science. In this way I tried to check my own opinions and beliefs and those of other people concerning important subjects. I found findings of science, e.g. that planets move according to the same laws as a stone thrown on earth, much more fascinating than miracles believed to be happening in Lourdes, a Roman Catholic place of pilgrimage. Besides, I found it suspicious that never a lost arm, leg or eye was restored, although this appeared no more difficult for an almighty god than the performance of the common kind of miracles which are practically impossible to assess as such. I will now discuss, how, at the end of the first year of my study at the university, I came to the conclusion that the selection and modification of a model, theory, or symbol system on the basis of experience is the only way, in which valid knowledge can be obtained. The main subjects of my undergraduate study were physics and mathematics. After reading a few books on philosophy, I was carried away by a reasoning of Kant in his work "Criticism of pure reason". He asked the question, how we can be certain that the Euclidian axiomata are valid in the real world. This cannot be based on experience, because experience does not provide certainty. Kant's answer was: the structure of the mind is such, that it cannot think in a different way. Soon, however, I learned that since Kant different mathemathetical geometries had been developed, logically as self-consistent as euclidian geometry. Any feeling of certainty about the applicability of a theory to the real world is apparently illusory. Only comparison with experience or experiments can decide this applicability. Another change in my convictions, now concerning time, was necessitated by Einstein's theory of special relativity. I was guided to the solution of the problem of knowledge by reading parts of David Hume's "A treatise of human nature" and "Enquiries concerning the human u n d er s tandi ng. . . " , to which my attention was dra~vn by the remark of Kant that this

philosopher had shaken him out of his dogmatic slumber. Recently I read that Einstein had stated that study of ideas of Hume had given him the courage to develop and publish relativity theory. It is rather remarkable that Einstein did not accept the experimentally supported statistical interpretation of quantum mechanics, because of his assumption that "God does not play dice". Because Einstein spent the last 25 years of his life trying to develop in vain a causal theory replacing quantum mechanics, this opinion was presumably a deeia conviction rather than a model or hypothesis to be replaced by another hypothesis fitting better with experience. This example and those given in the preceding paragraph indicate that untested convictions or beliefs are not a sound basis for science or for thinking in general. About one year before completing the study for the degree of "doctorandus" in physics and mathematics, I was appointed as assistant at the Biophysical Research Group, which was housed in the Physics Laboratory of the state University at Utrecht. The senior investigator in this group was Dr. E.C. Wassink, an internationally known research scientist in photosynthesis. Bessel Kok was completing his thesis research for his PhD thesis on the quantum yield of oxygen production of Chlorella. My task was to measure absorption spectra of purple bacteria for Wassink. By simplifying the operation of a home-built absorption spectrophotometer, I reduced the time required for this routine job from two half days to one or two hours per week, which gave me time for a little reading on photosynthesis. Already at high school, I was interested in photosynthesis, an almost unbelievable process, in which water and an extremely dilute atmospheric gas carbon dioxide with sunlight as source of energy were converted into the substrate of life. No one had imagined this process, before experimental facts demonstrated its existence. Our biology teacher even stated that we probably never would know, how photosynthesis occurred, because photosynthesis stopped as soon as plants were broken up. Not being hindered by much knowledge, I asserted that it probably would be possible to establish how photosynthesis occurred by just carrying out quantum mechanical calculations on various configurations of chlorophyll, water and carbon dioxide. Ten years later, Wassink showed me the formula of chlorophyll a, and said:

65 "look at this", and ten seconds later "you are a physicist, tell me how it works". I said that I thought that reactions between entities much more complicated than two hydrogen atoms were at present too difficult to establish by quantum mechanical calculation. Shortly before I obtained the doctorandus degree, which in the Netherlands is the formal end of the University study and has to be obtained before the PhD, Professor Milatz, who was the director of the Biophysical Research Group and of the Physics Laboratory, offered me a half time assistantship in this Group, but I had to do my PhD research on the remeasurement of Planck's constant c2. I insisted that I would work full time on a subject in photosynthesis. Although this was the first time that a physicist would in Utrecht work on a biophysical PhD subject, my condition was accepted after some hesitation, which enabled me to become a professional biophysicist.

making the simplest assumptions, and only changed these if forced by results of critically designed experiments. Thus I first imagined that in any given species, only one homogeneous pigment system was present, in which the pigment molecules were distributed as in a solution. I was, however, aware that, in biological systems, even a plausible hypothesis supported by quantitative experiments is far from certain, because of as yet unknown complicating factors. Later I used to urge my students to attempt to achieve accordance or consistency with their model or hypothesis by varying experimental procedures, but, when successful, to investigate critically whether errors were made or inconsistencies could be produced by other types of experiments. Extreme carefulness and self-criticism is in order, when conclusions are inconsistent with a theory supported by clear-cut experimental evidence of various kinds, as opposed to one only weakly supported experimentally, but accepted by the majority of scientists on basis of fashion or authority.

1.2. Research on transfer of electronic excitation energy in photosynthesis Wassink, who would soon be leaving being appointed as a professor of plant physiology at the Agricultural University at Wageningen, suggested as a PhD subject the study of the kinetics of chlorophyll a fluorescence. From such study evidence, presumably, could be obtained concerning the photochemical reaction, which was assumed to affect these kinetics. Wassink told me about the kinetic work in his and two other laboratories, which was interpreted differently in the three "schools". Since the kinetics appeared to reflect a number of unknown biophysical reactions, I chose as a physicist, knowing little about photosynthesis, to study the clear-cut problem of energy transfer, the possibly first step in the path of light energy. When I began research on energy transfer in photosynthesis, nothing was known about this subject, except that light energy absorbed by the carotenoid fucoxanthin in certain species of diatoms was transferred to chlorophyll a, but even this conclusion, although true, appeared to have been based on, in my opinion, incomplete and shaky evidence. In order to make a study of unknown possibly complicated mechanisms feasible, I started by

1.2.1. Light energy absorbed by various pigments is, in the form of electronic excitation energy, transferred to photosynthesis via ( bacterio )-chlorophyll By measuring excitation or action spectra of fluorescence of bacteriochlorophyll and chlorophyll a in photosynthesizing organisms, I had found that light energy absorbed by other pigments such as carotenoids was in whole or part transferred to the chlorophyllous pigments. In order to draw this conclusion, it was necessary to measure and analyze the emission spectra of a cell suspension in terms of the emission spectra of the fluorescing molecules present. When I started, no reliable emission spectra of photosynthetic organisms had been published. I found that the action spectra of (bacterio)chlorophyll fluorescence were roughly proportional to those of photosynthesis, which indicated that quanta were only active in photosynthesis if transferred to the fluorescing chlorophyll, which was chlorophyll a in algae and the long wave bacteriochlorophyll complex in purple bacteria.

1.2.2. Evidence for two pigment systems Shortly after it became available, I bought Volume

66 I of Rabinowitch's (1945) masterly monograph on Photosynthesis and related Processes. As far as I know, Rabinowitch was the first author (p. 162) that discussed clearly the hypothesis of two different primary processes in series. His scheme may be written as: H 2 --~ Z --*

(hv) ~ Y ~ (hv) ~ X ~ C O 2 (1)

in which the arrows represent hydrogen or electron transfers, and the hv's indicate photochemical transfers. In view of the available evidence, he considered the hypothesis of two different photochemical reactions as a second choice. Rabinowitch's scheme implied another hypothesis, namely that water oxidation is a dark reaction, requiring 4 redox equivalents of the primary oxidant Z per oxygen molecule produced. I had read Rabinowitch's discussion with great interest, and thus almost from the beginning of my research in photosynthesis, I was aware of the possibility of more than one photochemical reaction. As will be discussed, my studies on excitation energy transfer demonstrated the existence of more than one pigment system in a number of species, but this was, for good reasons to be discussed, not taken as evidence for two major photosynthetic systems. Direct strong evidence for such systems was only forthcoming a decade later, after a new method for studying photosynthetic intermediates had become available. | first studied energy transfer from carotenoids to bacteriochlorophyll in the nonsulfur purple bacterium Rhodospirillum rubrum. In this bacterium two carotenoids were present: rhodopin and spirilloxanthin, the characteristic three banded bluegreen absorption spectrum of the latter pigment being somewhat shifted to longer wave lengths. The action spectrum for photosynthesis, which had been measured in an indirect way, showed in addition to the bacteriochlorophyll band only the rhodopin bands. The fluorescence action spectra of the bacteria, which were grown in closed bottles, varied from culture to culture. After a long search I found that the absorption and fluorescence action spectra of "young" cultures showed the rhodopin bands, while those of "old" cultures showed the spirilloxanthin bands. Assuming, that only the "young" bacteria were observed in the photosynthesis spectra, I could maintain the hypothesis that photosynthesis proceeded via bacteriochlorophyll,

excited either directly by light absorption or indirectly by energy transfer from an "accessory pigment". Similar experiments with two other species of purple bacteria led to the same conclusion, without the complication of variable action spectra. In the green alga Chlorella and in a diatom, chlorophyll a was found to play a role analogous to that of bacteriochlorophyll in purple bacteria. Quanta absorbed by chlorophyll b in Chlorella were transferred in the form of excitation energy with close to 100 percent efficiency to chlorophyll a, and quanta absorbed by the carotenoid fucoxanthin in a diatom with about 80 percent efficiency. The following anomalies observed in red and blue-green algae suggested to me a decade later experiments which, against my expectation, demonstrated the existence of more than one photosynthetic system. French and Young (1952) and I (1951, 1952) had independently found that quanta absorbed in the red alga Porphyridium cruentum at the chlorophyll a maximum around 430 nm were less active in exciting chlorophyll a fluorescence than quanta absorbed by the major red pigment phycoerythrin at its maximum at 550 nm. French and Young explained this unexpectedly low chlorophyll a fluorescence upon 430 nm excitation by the assumption that carotenoids, which also absorb around 430nm, formed an opaque screen around the chlorophyll. I found, however, that quanta absorbed at 670 nm, where only chlorophyll a shows absorption, had the same low yield for chlorophyll a excitation. My conclusion was that two forms of chlorophyll were present, a fluorescent form, to which quanta absorbed by phycoerythrin were transferred with good efficiency, and a non- or weakly-fluorescent form, which substantially contributed to the absorption spectrum, but not to the fluorescence excitation spectrum. Similar conclusions were obtained also for phycocyanin in various species of red algae and blue green algae (now called cyanobacteria). When an undergraduate student, Joop Goedheer, and I planned to measure algal photosynthesis action spectra, a publication by Haxo and Blinks (1950) appeared, describing a simple but rapid and very sensitive polarograph. They had measured action spectra and the rate of oxygen evolution of a number of red algae, which

67 showed that quanta absorbed by chlorophyll a were less effective in photosynthesis than quanta absorbed by phycoerythrin. Haxo and Blinks suggested that phycoerythrin participated in photosynthesis independent of chlorophyll. I had used different species for fluorescence measurements. Thanks to their example, we were able to construct in a few days a similar polarograph, and to determine photosynthesis action spectra in a much shorter time than first expected. We found that the phycobilins also in the species I had used in the energy transfer experiments were more effective in photosynthesis than chlorophyll a. My interpretation, which soon was also accepted by Larry Blinks, was that also in red and blue green algae photosynthesis proceeded via chlorophyll a excitation; the non- or weakly-fluorescent form of chlorophyll was assumed to be not or less active in photosynthesis.

1.3. Looking under the hood When it had become clear that excitation energy was transferred to photosynthesis via (bacterio) chlorophyll, I began thinking about a method for studying the photochemical events following the excitation of these chlorophyllous pigments. I reasoned that if chlorophyll participated directly in the photochemical reaction, its absorption spectrum would presumably change, like that of other pigments upon oxidation or reduction. The possible absorption changes, occurring in photosynthesizing cells upon illumination, should be small, since otherwise they would have been discovered already by the naked eye, which is rather sensitive to color changes. Excitation energy might be transferred to a fraction of the (bacterio)chlorophyll or to another pigment present at a low concentration. This hypothetical pigment, which I called P, or reaction center (Duysens, 1952, pp. 88-90), was assumed to be photochemically active and to change its absorption spectrum upon illumination. According to F6rster's (1948) theory of excitation transfer, efficient transfer is only possible if the energy receiving molecule is a pigment with an absorption band overlapping the fluorescence band of the energy transferring molecule. An absorption band of P should thus occur at the same or slightly longer

wavelength than that of the longest wavelength band of (bacterio)chlorophyll. The bulk of the bacteriochlorophyll, which I called energy transferring bacteriochlorophyU and was later by another author called antenna bacteriochlorophyll, was assumed to be photochemically inactive. Applying Fbrster's theory, I estimated that in purple bacteria P should be present at a concentration of about one percent of that of bacteriochlorophyll, in order to make efficient trapping of excitation energy by P theoretically possible. Using parts of the homebuilt fluorescence spectrophotometer, we constructed an absorption difference spectrophotometer sufficiently sensitive to measure changes of this order of magnitude. I thought that the absorption band of P might well be bleached upon photochemical conversion of P. Since such a bleaching would eliminate P as a trap, an increase in (bacterio)chlorophyll fluorescence yield would occur upon illumination, provided bleached P would accumulate and not be reconverted to P by very rapid dark reactions. Since Wassink et al. (1942) had observed a large increase in fluorescence yield upon illumination of purple bacteria, species from the two sub-groups of this group of bacteria were used for our experiments. Surprisingly easily the experiments yielded a result consistent with the discussed hypothesis: a decrease of an absorption band at 890 nm. This part of the spectrum indicated that at saturating intensity about 3 percent of the bacteriochlorophyll molecules was bleached, indicating that a small part of the bacteriochlorophyU functioned as a reaction center. In my thesis, however, I considered this conclusion not the most probable one, because in the purple bacterium Rhodospirillum the concomitant bleaching of a band at 800 nm could, in the purple bacterium Rhodospirillum, not be attributed to the one spectral form of bacteriochlorophyll that I believed to be present. When a few years later independent evidence was obtained for the existence of another spectral form with a maximum at 800 nm, we reinterpreted the experiments in terms of the reaction center hypothesis (Duysens et aL 1956); the short wave-length bleaching could be attributed to an indirect effect of the photooxidation of the reaction center bacteriochlorophyll, which caused the bleaching of the long wave length band. Rabinowitch later said in a lecture that, for the

68 study of photosynthesis, absorption difference spectroscopy was comparable to looking under the hood of a car in order to find out about its mechanism, as compared to studying its gas exchanges, which was the earlier method of choice in photosynthesis. When I almost had completed my doctoral thesis, Dr. C.S. (Stacy) French, director of the Department of Plant Biology of the Carnegie Institution of Washington at the Stanford campus (Palo Alto, California) visited our laboratory. After I had told him about my experiments, he invited me to work for a year as a Carnegie fellow in his laboratory, and to attend, on the way from New York to Palo Alto, a photosynthesis conference at Gatlinburg (Tennessee) organized by Eugene Rabinowitch, Hans Gaffron and himself and to take place in October 1952. I gratefully accepted both invitations. The conference was attended by mtast senior scientists active in the field. I sent a copy of my thesis, which had been printed shortly before the meeting, to each of the (less than hundred) participants. At the meeting Rabinowitch gave me ample time to present my results in the session he chaired. Dr. James Franck, a physicist (nobel laureate), who had switched to photosynthesis, remarked in the discussion that the light induced absorption changes around the bacteriochlorophyll absorption bands in purple bacteria were small and were in his opinion artifacts. I gave some technical arguments in favor of the reality of the changes, but added that I was uncertain about the interpretation of the phenomena. It may be noted that Franck thought that the excitation energy could not be transferred over more than 10 chlorophyll molecules and also that each chlorophyll molecule was reactive (see Duysens 1952, 1986). Most senior people in photosynthesis knew now about my work on energy transfer or at least had acquired the belief that photosynthesis proceeded after the transfer of the excitation energy to chlorophyll. After arrival at his laboratory, Dr. French suggested that I should continue work on energy transfer, using his ingeniously constructed automatic fluorescence spectrophotometer. This apparatus would greatly have facilitated my PhD research on energy transfer. When I said that I had become more interested in finding out what happened after

chlorophyll excitation and that I therefore preferred to study possible intermediates in purple bacteria and oxygenic organisms by means of absorption difference spectroscopy, he told me that after hearing in Utrecht about the absorption changes in purple bacteria, he and Britton Chance tried in vain to find light-induced absorption changes in Chlorella, using Chance's apparatus developed for studying the light induced dissociation of the cytochrome oxidase-CO complex. He also said that he did not have an absorption difference spectrophotometer. I said I could build a simple one myself in a short time, at least sufficient for studies on purple bacteria. After some hesitation, Stacy generously allowed me to give it a try, advised and even helped me with his own hands by turning an important part on a lathe. He kept me also from entering side paths by strongly dissuading me from attending Van Niel's course on photosynthetic bacteria, and from using a Warburg apparatus in order to check photosynthetic bacterial activity. Within a few months a sufficiently sensitive apparatus could be put together, consisting of a small monochromator, simple optical and electronic components and a Brown recorder. When, after about one year, I moved to Eugene Rabinowitch's Laboratory at the University of Illinois at Urbana, Stacy kindly allowed me to take the home made parts of my apparatus. In the following section, the experiments at the two laboratories are described which formed a prelude to the discovery of the two photosynthetic systems.

1.4. Cytochromes and reaction centers in purple bacteria First I studied the infrared absorption changes, possibly connected with the reaction center in Rhodospirillum rubrum. Since the light-induced bleaching occurred more readily in the presence of air and in the absence of a hydrogen donor, this suggested to me that the bleaching was a photooxidation. In the presence of hydrogen donor, higher actinic intensities were required for the same amplitudes of steady state bleaching. Later it was shown (Duysens, 1959) that a bleaching, spectrally similar to that in light (Goedheer, 1959), occurred in the dark by exposing bacterial chromatophores during one minute to 0.01 M ferricyanide, which

69 was reversed within a few seconds by adding 0.023 M ferrocyanide. The redox potential for half bleaching was between 0.40 and 0.47 volt. Next I scanned the visible region for absorption changes. At low actinic intensity and in the presence of a hydrogen donor, a light minus dark difference spectrum was found with unusual sharp peaks, one at 428 and a smaller one at 550 nm. In one of the text books in the Biochemistry Library of Stanford University, I found, to my satisfaction and surprise, a strikingly similar spectrum, obtained by H. Theorell by subtracting the spectra of oxidized and reduced cytochrome c. Only the 428 nm peak was shifted to shorter wavelengths. I concluded that in Rhodospirillum a cytochrome was oxidized upon illumination and reduced in the dark. i was surprised, because at that time there was no reasoia to assume that cytochromes played a role in photos~cnthesis. Davenport and Hill (1952) had extracted cytochrome f from chloroplasts. I do not remember whether I saw this paper. If I did, the mere presence of a substance in chloroplasts would for me not have been a reason to assume that it played a role in photosynthesis. Photosynthetic phosphorylation had not yet been discovered, and it was quite possible that other processes, such as respiration, occurred in the chloroplasts in addition to oxygen evolution and the light reactions.

1.5. Cytochromes and pyridine nucleotide in algae After my stay at the Carnegie Institution, Stanford, I spent another year in the USA with Professor Eugene Rabinowitch at the Photosynthesis Group of the Department of Botany at the University of Illinois at Urbana. Through his excellent monograph (Rabinowitch 1945, 1951), he had been my main teacher of photosynthesis. Another member of this group was Professor Robert Emerson, who had made important experimental contributions to this field. The paper of Emerson and Lewis (1943) on photosynthesis action spectra had been for me an admirable example of biological spectroscopy. Shortly after my arrival Rabinowitch asked me wether I really believed that the weakly-fluorescent chlorophyll a in red and blue algae, called inactive in my thesis, was indeed biologically inactive. When I said that this seemed unlikely because of

evolutionary pressure or teleology for short, he asked me to investigate this, but since we could not devise promising experiments, which could be carried out in a short time, it was decided that, instead, I would attempt to study the electron transfer chain in algae by characterizing electron transfer components and studying their kinetics by means of absorption difference spectroscopy. I had at my disposal a simple but powerful Bausch and Lomb monochromator, which combined with the parts brought from Dr French's laboratory, resulted in an absorption difference spectrophotometer of superior signal-to-noise ratio and usable down to 330 nm. In French's laboratory, I had observed in the light minus dark spectrum of the green alga Chlorella small negative peaks at 555 and 420 nm, which had been tentatively attributed to the oxidation of cytochrome f (Duysens, 1954b), but this attribution was uncertain because of the presence of a much larger positive band at 520 nm and a negative band at 480 nm. However, in the red alga Porphyridium cruentum, a "pure" cytochrome difference spectrum was observed (Duysens, 1955b), similar to the redox difference spectrum of cytochrome f, extracted from higher plants (Davenport and Hill, 1952). Our observations thus showed that cytochrome f or a spectrally similar cytochrome was oxidized upon illumination. The kinetics upon illumination indicated a quantum yield sufficiently high to be consistent with a major role of this cytochrome in algal photosynthesis. An increase in absorption around 360 nm was attributed to light-induced NAD(P) reduction, but the band was too broad for a definite identification. With chloroplast preparations, photoreduction of added NADP had earlier been demonstrated by reactions which rapidly removed the NAD(P)H formed.

1.6. Complicated kinetics of cytochromes and absorption changes at 515 nm After my return to Utrecht, I worked out and tried to interpret some of the data obtained in the USA. I reported on this work in October 1955 at the second Gatlinburg Conference (Duysens, 1957) and at the third international biochemical congress (Duysens, 1955a). Within three years after my

70 demonstration that significant reversible lightinduced absorption kinetics and absorption difference spectra could be measured in photosynthesizing cell suspensions, there were, at this conference, already contributions from the laboratories of Chance, French, Rabinowitch, and H.T. Witt, based on this type of measurement. In other laboratories sensitive absorption difference spectrophotometers were being adapted or constructed. People may wonder now, why this powerful technique had not been used before. The main reason probably is that the earlier attempts yielded negative results or artifacts, because of insufficient sensitivity and/or improper methods for excitation and measurement. My attempt was successful, because I had, by applying a specific model for photosynthetic energy transfer and trapping to my data, an idea of the possible size of the photosynthetic unit and thus of the sensitivity required, of the most promising spectral region and organisms and of the actinic intensity needed. Without this information, I might not have taken the risk of wasting my time by searching blindly for absorption changes nobody had ever seen. I remember that, when I visited Oak Ridge National Laboratory in 1955, the Deputy Director Dr S.F. Carson of the Biology Division asked me to estimate how long it would take before the main intermediates and reactions of the photosynthetic electron transfer chain would be known. I thought that major progress would be achieved within five years. It took a much longer time than I expected, because of unforeseen complexities in the photosynthetic apparatus. This may be exemplified by light-induced cytochrome reactions in purple bacteria (Duysens, 1954a). By absorption difference spectroscopy it had been observed, both by Chance and coworkers and by me, that more than one cytochrome species is oxidized upon illumination. In "an attempt to explain the various observations" (Duysens 1957) a model was presented, which may be described as follows. Upon illumination a small fraction of the bacteriochlorophyll is photooxidized and simultaneously a reduced compound H is formed. This oxidized bacteriochlorophyll oxidizes one or more reduced cytochromes. A fraction of these cytochromes oxidizes the bacterial hydrogen donor, the remaining fraction reoxidizes part of H, generating ATP. The remaining H reduces carbon dioxide with the aid of the ATP.

Although the general aspects of this model are presumably correct, extensive studies in several laboratories were required to identify the cytochromes and additional redox intermediates and to develop much more sophisticated models to explain the observed kinetics. The cytochromes and other redox components function partly in cycles playing a role in proton transport and the generation of ATP. These studies required not five years but several decades of research. Certain phenomena are still unexplained. Also difficult, at least initially, was the interpretation of the light induced absorption increase at 515 nm observed in Chlorella and higher plants (Duysens, 1954b). This phenomenon was studied by various laboratories and shown to be associated with a number of reactions. Since the 515 nm kinetics appeared to depend on presumably indirect effects of the reactions of a number of largely unknown compounds, I decided, when I returned to the Netherlands in 1954, to concentrate research on more directly observable intermediates with presumably simple kinetics. One of these intermediates was NAD(P).

1.7. Photosynthetic pyridine nucleotide reduction, phosphorylation, and quantum requirements The writing of a review on photosynthetic energy transformations forced me to think about the thermodynamics of this process (Duysens, 1956). In a scheme in this review the free energy of the redox couples was plotted vertically. In principle, this scheme was similar to that discussed above for purple bacteria: by one quantum strongly oxidizing and reducing molecules Z and HX were formed by light driven hydrogen or electron transport. "Part of the HX and Z is short-circuited via cytochrome f a n d via some other couples of intermediate oxidation reduction potential... The main part reduces PN + (NADP); the corresponding Z dehydrogenates water to give oxygen." The scheme also showed, that the free energy difference between HX and NADP was sufficient for the phosphorylation of 1 ADP molecule per two equivalents. Such phosphorylation could explain the so called non-cyclic photophosphorylation which is coupled to NADPH and oxygen production (Arnon, 1958). It was not necessary to assume, as Hill and Bendall

71 later (1960) did, two photochemical reactions in series to explain this phenomenon (see section 2.3). A scheme as described would require at least four quanta for the reduction of one carbon dioxide molecule, while a scheme with two reactions in series would require at least eight quanta, if it is assumed, as I proposed in my thesis, that one quantum is needed for the production of one reducing equivalent in a photochemical reaction. After discussing several papers on quantum requirements (Duysens, 1956), I concluded "In spite of discrepancies between the results of several laboratories, the recent determinations of the quantum requirement of Chlorella... seem to indicate that it is in the range 4 to 8." Notably Bracket et al. (1953) had reported quantum requirements of 6.1 + 0.6, which I thought were reliable. In view of the evidence available at that time, Hill and Bendali's later proposal of two reactions in series was an unlikely hypothesis. Since the broad 360 nm absorption band, which I had attributed to NADPH, appeared insufficiently specific for reliable kinetic measurements, I developed together with Jan Amesz a new method, based on fluorescence spectroscopy, by means of which NAD(P) redox reactions could be measured. Jan did the six months experimental work for a physics minor in chemistry in my laboratory. The validity of the method was demonstrated in yeast cells (Duysens and Amesz 1957): upon addition of glucose to starved aerobic cells, the fluorescence at 450 nm, emitted by N A D H but not by NAD upon excitation with 366 nm light from a mercury lamp, more than doubled within three minutes. We remarked "The approach to the .. steady state is .. a damped oscillation with a period of about one minute." and also that the fluorescence method "in contradistinction to that of absorption photometry, can also be used for measuring reduced pyridine nucleotide in the surface layer of large intact organisms or organs such as large muscles." Our paper was followed by many papers from Britton Chance's and other laboratories, on fluorescence studies of NADP reactions in non-photosynthetic cell suspensions and organs such as brain and heart. This again shows that the demonstration of the feasibility of a method plays an important role in the progress of science. In cooperation with other students, the first evidence for NAD(P) reduction in intact purple bac-

teria was obtained by measuring a light-induced increase in a fluorescent band around 430 nm. In 1958, Jan Amesz started his PhD research on nicotinamide-dinucleotide reactions in photosynthetic organisms. At about the same time, John Olson, who had just obtained the PhD degree with Britton Chance on a study of cytochrome reactions in the sulfur purple bacterium Chromatiurn, started work in our laboratory. John concluded his thesis with the statement that NAD(P) was probably not reduced upon illumination of photosynthetic cells. Together with Jan Amesz, he showed by means of fluorescence spectroscopy that the opposite was true, at least for purple bacteria. Jan Amesz also studied the light-driven reduction of NAD(P) in algae and, of added NADP in chloroplast preparations and measured the quantum requirement for this process. This work was reported at the 1958 Brookhaven symposium (Duysens, 1959). We had estimated that the number of quanta required per 4 reduced equivalents was not more than 6. However, in an erratum it was stated that a calibration error had been made and that this number had to be increased to 12. If indeed less than 8 quanta per 4 equivalents would have been sufficient, it would have been impossible that two one quantum reactions cooperated to transport one reducing equivalent. In 1958 there was no convincing indication that two major photosynthetic reactions might be cooperating in oxygenic photosynthesis, but this changed in the following years.

2. Discovery of the two photosystems 2.1. Miscellaneous observations indicating the existence of several photochemical reactions: Chromatic transients and enhancement effects At the second Gatlinburg conference in October 1955, Blinks (1957) had reported that in red algae, illuminated alternately with red and green light for a few minutes, qualitatively different photosynthetic transients occurred, which were not caused by differences in intensity. Robert Emerson suggested in the published discussion (p. 448) that these effects may be due to a light induced change in respiration (different for green and blue light). The evidence given by Blinks indicated, I thought, that

72 the chromatic transients were not artifacts. This implied that these transients were caused by two or more photochemical reactions with different action spectra. The effects seemed small and could be explained by the assumption that the activity of the one photosynthetic reaction or of respiration was changed by one or more other light reactions. Several light-induced processes different from photosynthesis were known, such as the redinfrared effect. Govindjee et al. (1960) observed that the steady state chlorophyll a fluorescence of Chlorella upon excitation with combined light beams of 436 and 700 nm was smaller than the sum of the fluorescence intensities in the separate beams. They suggested that "Perhaps, excitation at 700 nm generates an energy sink;". Kautsky et al. (1960) observed rapid changes in fluorescence yield during light flashes, which were explained by two light reactions, alternatingly driven by one photoactive chlorophyll but having opposing effects on fluorescence yield. Although this was a plausibl¢ explanation, and proved to be partly correct, it was not a useful starting point for further research, because the two proposed reactions were incorrectly attributed to one photochemical system. Results obtained by Emerson and coworkers induced me to approach the problem of the two wavelengths effects by means of absorption difference spectroscopy. Emerson et al. (1957; see pp. 141 and 142) tentatively concluded from experiments on the red alga Porphyridium cruentum that the low quantum yield of photosynthesis for light, absorbed mainly by chlorophyll a at 680 nm, could be enhanced to the same high yield as that of shorter wavelength light, mainly absorbed by the phycobilins, by simultaneous illumination with short wavelength light. Emerson and Chalmers (1958) speculated that accessory pigments played some specific role in photosynthesis, but this was unlikely in view of the efficient energy transfer from these pigments to fluorescent chlorophyll a, discussed in the introduction. I thought that the "inactive" weakly fluorescent chlorophyll might participate in photosynthesis after being activated by a side reaction of the photosynthetic reaction driven by the fluorescent chlorophyll a. In order to solve this question, I decided to measure action spectra for cytochrome oxidation and NADP reduction in Porphyridium in the presence and absence of short and long wavelength background illumination.

This proved to be the experimental handle to approach the problem posed to me by Rabinowitch, when I arrived at his laboratory half a dozen years earlier, about the physiological function of the so called inactive chlorophyll a (section 1.5.). The difference was that we now had identified and could measure two important photosynthetic intermediates in Porphyridium, a species with a lot of "inactive" chlorophyll a, and that Emerson's enhancement experiments indicated that this chlorophyll could be "activated" to high photosynthetic efficiency.

2.2. Experimental establishment of the existence of two photosynthetic systems with separate reaction centers Before the set up for simultaneous background illumination with two colors could be used, the measurements of cytochrome oxidation in green or red light yielded surprising results, which were reported at the 1960 international photobiology congress (Duysens, 1961). Contrary to my expectation of a high oxidation yield, a low yield was found at 560 nm, where the photosYnthetic yield was high, and a high oxidation yield was found at 680nm. The action spectrum for cytochrome oxidation showed a large peak at 680nm caused by chlorophyll a and small bands at about 630 and 560 nm, due to phycocyanin and phycoerythrin. In order to explain these and my earlier experiments and Emerson's enhancement effect, I postulated the existence of two major photosystems, 1 and 2. System 1 contained the weakly fluorescent chlorophyll a, formerly said to be inactive, and oxidized cytochrome; system 2 contained the fluorescent chlorophyll a. An interaction between the two systems was shown by the different kinetics of cytochrome oxidation at different actinic wavelengths. The paper ended with the remark: "Because of the spatial separation of the photochemical systems, suggested by the preferential transfer of energy from phycobilins to chlorophyll a2, isolation by extraction might be feasible with the absorption changes as a guide." In the discussion following the paper, C.P. Whittingham asked me whether my experiments supported the scheme proposed by Hill and Bendall in Nature. I said that I did not know, I had not seen

73 the paper. After returning to the laboratory, we established that cytochrome oxidized by 680nm background, mainly exciting system 1, was reduced by superimposing 560 nm light, mainly exciting system 2. ! remember that the modification of the actinic beams optics had been completed in the afternoon before I left for the Copenhagen meeting, but that the central battery needed for a xenon lamp could not be switched on, because the attendant in charge had left the laboratory, taking the key of the switching room. Since the photoreduction and oxidation of cytochrome occurred with a reasonable quantum yield, these experiments indicated that the 2 systems acted in series to produce complete photosynthesis. It was also established that DCMU, 3-(3,4dichlorophenyl)-l,l-dimethylurea, and some other chemicals inhibited system 2 (cytochrome reduction), but not system 1 (cytochrome oxidation). By inhibiting system 2 by DCMU the action spectrum of cytochrome oxidation by system 1 was determined. This action spectrum was similar to that of NADP reduction in intact cells, indicating that this reduction was also driven by system 1. The series hypothesis implied that not only cytochromefbut all other redox intermediates present in a linear electron transport chain between the two systems, would become oxidized in light 1, which is light of a wavelength band mainly absorbed by system 1, and reduced in light 2. These experiments were published in a article (Duysens et al., 1961), which ended with the remark: "A more detailed report is in preparation, in which we will discuss partly similar but less detailed and experimentally less supported hypotheses concerning the role of the photosynthetic pigments which have been proposed recently by other authors." Before we did the experiments described in the preceding section, I preferred the hypothesis of a single major primary photoreaction, because the two wave-length effects could plausibly be explained by ad hoc hypotheses, and because there were determinations of quantum requirements smaller than 8, which were inconsistent with a scheme requiring two one quantum reactions per redox equivalent transported from water to carbon dioxide. Since these determinations were difficult and based on plausible but uncertain assumptions, preference switched to the hypothesis of two major photochemical systems in series, when it was estab-

lished that cytochrome f was alternately oxidized and reduced with good efficiency by switching between two actinic wavelengths.

2.3. Speculations and investigations in other laboratories. Confirmation and extension Robin Hill had worked in the laboratory of David Keilin, who had established the role of cytochromes in respiration. Hill and coworkers had been searching for the presence of cytochromes in chloroplasts and discovered two cytochromes, called f and b 6. Davenport and Hill (1952) measured the midpoint potential of cytochrome f and estimated that the free energy required for the reduction of 4 equivalents of cytochrome f and the simultaneous formation of one mole oxygen was 173 k J, which was almost identical to "the value of the light energy per Einstein for the maximum of the fluorescence band of chlorophyll a in the plant at about 20°C '', which was estimated to be 174 kJ. The authors also noted that the midpoint potentials between cytochrome f, cytochrome b 6 and the carbon dioxide reducing entity are separated by similar potential differences of about 0.4 volts. I have difficulty pin-pointing the meaning of their tentative, multi-interpretable and often conditional discussion statements, but, as suggested by remarks like "we are almost obliged to conclude that these energy relations are significant and not fortuitous', the authors appeared to imply that three one-quantum reactions occur in series, each transferring 4 electrons. This would mean a roughly 100 percent conversion of excitation energy to free chemical energy and a quantum requirement of 3 per molecule for the production of oxygen or uptake of carbon dioxide, in accordance with results reported shortly before by Otto Warburg and coworkers (see Rabinowitch 1956), but not quoted by the authors. Finally, in their famous paper, Hill and Bendall (1960), after quoting Davenport and Hill's paper, first discuss a potential diagram showing three light reactions between water, cytochromef, b6, and carbon dioxide and remark "The postulation of two light driven steps, rather than three, would be in better accord with present experimental results. In this case oxidized cytochrome b 6 would have to be reduced by YH to give YOH (the oxidant for oxygen) and cytochrome f would have to be oxidized

74 by X to give XH (the reductant for carbon dioxide)•" Hill and Bendall assumed, contrary to Davenport and Hill, that one redox equivalent is moved per photoact, as I did in my thesis• The new scheme was consistent with the observation (Duysens 1955b) that cytochrome f is oxidized upon illumination, but this was not noted• Hill and Bendall also postulated that non-cyclic phosphorylation occurred between the two cytochromes, which explained the coupling between phosphorylation and NADPH production• They did not discuss the possibility that cytochrome could be involved in cyclic phosphorylation and that non-cyclic phosphorylation might occur at one of the ends of the electron transfer chain. Neither did they mention Emerson's enhancement effects (Emerson et al. 1957)• Instead they remarked: "If the reaction bet w e e n f a n d b 6 could be inhibited, we would expect •.. thatfwould become oxidized and that b6 would tend to be reduced in light•" and made the puzzling suggestion that this might be studied in purple bacteria• When writing the present review article, I noted that Rabinowitch already in 1956, p. 1862, interpreted the light induced cytochrome f oxidation in Porphyridium cruentum, which I observed in his laboratory, in two possible ways. One was similar to that proposed by Hill and Bendall in 1960. He also stated: "The quantum requirement of the hydrogen transfer reaction as a whole would be (at least) 8, since two quanta will be needed to transfer each of the four required H atoms (or electrons), first from water to the cytochrome, and then from the cytochrome to the final acceptor." As a second possible hypothesis he mentions that of the cytochrome on a cyclic path between NADPH and oxygen or their precursors, both precursors being generated by a single photoreaction. If the kinetics ofcytochrome oxidation in Porphyridium cruentum had been measured between 1956 and 1960 both for excitation at 680 and 560nm and Rabinowitch's discussion had not been overlooked, Rabinowitch would presumably have been recognized as the "inventor" of the two photochemical systems in series. Hill and Bendall's article appeared before our results were submitted, but the initiation of our experiments was, as discussed, independent of this article• After reading it, which we did after completing most of the experiments related to cyto-

chrome oxidation, we looked more carefully and again in vain for the possible participation of cytochrome b 6 in a light driven redox reaction (see below). Kok (1959) first demonstrated antagonistic effects of two wave lengths in the blue green alga Anacystis. He found that light of about 680nm (light 1 in this species) caused a bleaching of a band at 700 nm and that the bleaching was reversed by light of 630 nm (light 2). He concludes that excitation generated by absorption at 680 nm is transferred to and bleaches "the 700 nm pigment"; excitation of the accessory pigment, phycocyanin, is transferred to this bleached pigment and regenerates the 700 nm pigment• This work was expanded by Kok and Hoch (1961) and was reported in 1960 in a symposium entitled Life and Light. They found that the 700 nm pigment could be bleached by mixtures of ferri- and ferrocyanide and had a midpoint redox potential of about 0.46 volt, which is of the same order of magnitude as that of the reaction center P in purple bacteria, which had been determined in a similar way (Duysens 1959). Making analogous assumptions as I had made about the reaction center in purple bacteria, Kok and Hoch concluded that the light induced redox reactions occurred with high efficiency. They named the 700 nm pigment P700, and proposed (see their Fig. 13) that P700 was oxidized by the first light reaction and, after dark steps, which were inhibited by DMCU, was reduced by a second light reaction. The first reaction produced XH, the second oxygen. Note that in Kok's (1959) and Kok and Hoch's (1961) schemes only one reaction center, P700, occurs, trapping two light quanta in succession, not two different reaction centers acting in series, as we had proposed• I only realized that when recently I read Kok's 1959 paper, which Govindjee kindly sent me. We did not consider a scheme with only one reaction center for interpreting the antagonistic effects of two actinic wavelengths on the redox states of cytochrome, because the two chlorophyll a containing pigment systems in red and blue green algae had to be spatially separate (Duysens 1952), which fitted, in contrast with Kok's scheme, in a simple way with the scheme of two spatially separate one-quantum reaction centers, each receiving excitation energy from one of the pigment systems• Also some properties of the photoreductant Q of the second

75 photoreaction, discussed in the next section, were inconsistent with Kok's scheme. Since Kok's data fitted in the series scheme and the relation between P700 and cy to ch r om e f i n algae was similar to that of P and cytochrome in purple bacteria, we assumed that his scheme was essentially the same as ours and did not attempt to understand the details of what seemed to me a rather clumsy and overcautious discussion. In a later paper, Kok et al., 1963, stated (p. 387) " I f one assumes two photoacts in photosynthesis, both sensitized by chlorophyll a, and if in both acts energy migrates by resonance transfer, two traps absorbing at long wavelengths ought to be present. P700, which we feel is one of these traps, bleaches upon excitation and therefore is amenable to spectroscopic investigation." The authors presented a scheme on a potential scale with two reaction centers P and Q in series (p. 393). Presumably because they could not find absorption changes attributable to the "hypothetical second sink", they left also open the possibility of one reaction center: " . . . P + . . . entering - or subject tothe second photoreaction." After Emerson's tragic death in an aircraft accident in February 1959, Rabinowitch submitted a paper (Emerson and Rabinowitch 1960), in which he interpreted partly unpublished results obtained by Emerson and coworkers on enhancement of photosynthesis by combining beams of two wavelengths. Also on the basis of my conclusion concerning the two types of chlorophyll a, he states: "A solution can be sought in the assumption of (at least) two different forms of chlorophyll a, which need to be excited to produce photosynthesis." Rabinowitch did not relate this with his first above quoted hypothesis concerning the two light reactions in series, connected by cytochrome f. This assumption of the two different forms of chlorophyll a was supported by the action spectrum of the enhancement of photosynthesis in light of about 690nm (Govindjee and Rabinowitch 1960). Although such action spectrum is a distorted spectrum of system 2 (Duysens and Amesz 1962), the very pronounced maximum at 670 nm in the diatom Navicula indicated that chlorophyll a was present also in this system. Our experiments provided evidence for the postulated role of cytochrome f, but not for that of c y t o c h r o m e b6, since the difference spectrum upon illumination with light 1 or 2 was similar to that of

cytochrome f (Duysens and Amesz 1962), indicating that cytochrome b6 was not in the linear electron transfer chain between the two systems. Later evidence has been obtained indicating that cytochr o m e b 6 may participate in a side cycle, which is driven by the electron transport chain between systems 1 and 2 (see e.g., Joliot and Joliot 1986). We also introduced for the first time correct methods for measuring the action spectra of the two photosynthetic systems (Duysens and Amesz 1962); these spectra are necessary for quantitative analysis, specifically for determining quantum requirements.

2.4. Kinetics of chlorophyll fluorescence in oxygenic organisms; The effect of the two light reaction on the acceptor Q of system 2 Believing that the complicated kinetics of the fluorescence of chlorophyll a, which had baffled so many experimentalists, were primarily caused by the interaction of the two photochemical systems, I thought that it would now be possible to tackle the task suggested by Wassink a dozen years before, when I started my thesis: to use this kinetics as a means for studying the mechanism of photosynthesis. An apparatus was constructed, which essentially recorded the fluorescence yield of chlorophyll a2, independent of the fluorescence caused by additional strong actinic beams of light 1 or 2; these beams were applied to shift the redox states of intermediates, which might affect the chlorophyll a2 fluorescence yield. A fixed weak measuring beam was modulated by interrupting it by means of a rotating disc with holes. Only the modulated fluorescence emission was measured by the use of a lock-in amplifier, not the stronger unmodulated fluorescence caused by the actinic beams. It took only a very short time to show that alternating actinic light 2 and 1 rapidly increased and decreased the chlorophyll a fluorescence yield. This could simply be explained by the assumption that a redox intermediate in the electron transfer chain between the two systems quenched the chlorophyll a 2 fluorescence in the oxidized, but not in the reduced state (Duysens and Sweers 1963). Although in short-time experiments fluorescence yield and reduction state of cytochrome moved

76 roughly parallel in alternating light 1 and 2, the hypothetical redox intermediate was different from cytochrome f, because in the dark (or in weak measuring light) the cytochrome was in the reduced state, but the fluorescence yield was low, implying that the intermediate was in the oxidized state. In preliminary experiments with DCMU, a high fluorescence yield was always found, also when the measuring light alone was on. However, after DCMU was added in complete darkness and then weak measuring light was switched on, the fluorescence was found to rise from a low level. Only one quantum per 150 chlorophyll a2 molecules sufficed for an appreciable fluorescence increase. The intermediate was called Q for two reasons: because Q (it is now called QA by most authors) quenched fluorescence in the oxidized state, and because quinone, specifically plastoquinone, was thought to be a possible candidate (see Duysens and Amesz 1962, p.257). Since the concentration of Q was of the same order as that expected for reaction centers, and its redox state strongly affected the fluorescence yield of system 2, Q was thought to be part of the energy trap or reaction center of system 2; one DCMU molecule attached to the reaction center prevented the oxidation of reduced Q by system 1, but not its reduction by system 2.

2.5. Subsequent developments The preceding section concludes this account of the discovery of the two photosynthetic systems. In the following decades, most of these conclusions have been verified and extended, the trapping pigment of reaction center 2, P680, analogous to P700, and many additional intermediates, reactions, and regulatory processes, such as a redistribution of pigments between the two photosynthetic systems upon changing the color of actinic light, have been established in a large number of laboratories by means of absorption difference spectroscopy and other methods. This required a much larger number of man-years than the experiments described, and still much work has to be done. These investigations not only greatly extended our knowledge, but were also necessary for checking and modifying the original results and conclusions.

3. Concluding remarks 3.1. Could the discovery of the two photochemical systems have been made earlier or later or in a different way? In his interesting short history of ideas in photosynthesis, Jack Myers (1974) states that until 1960 only a single kind of photochemical event had been assumed. "That was the simplest possible assumption. Where we had erred was in not recognizing, leaving tacit, that assumption." It is possible that many investigators were not aware of this assumption, but as I mentioned earlier, Rabinowitch (1945; also see 1956, p. 1862) had discussed a scheme (1), which was based on two light driven redox reactions. As mentioned, I was well aware of this scheme and, when the experimental evidence made it the most probable one, used Rabinowitch's symbols for the electron donor Z of system 2 and acceptor X of system 1, but not for the intermediate redox compound Y, because a "known" intermediate, such as cytochrome f, could take its place. The two system hypothesis was rapidly accepted, because it proved to be experimentally fruitful: after action spectra for the two systems were determined, Emerson's enhancement effects, and Blink's chromatic transients could be qualitatively explained, and the changes in absorption and fluorescence occurring within a few seconds could be explained and used for analysis. After treatment with detergents, two different particles, called system 1 and system 2 particles, were obtained by separation methods, containing reaction centers 1 and 2, and having the approximate pigment distribution and other properties as to be expected. There were details which could not be explained quantitatively and required additional assumptions e.g. about slow regulatory adjustments and unknown reactions and intermediates. These assumptioris have been partly confirmed. Although most experimental evidence is consistent with the hypothesis of two photoreactions in series as discussed, one photochemical reaction requiring only 4 quanta per oxygen and carbon dioxide molecule would thermodynamically be possible at not too low intensities (Duysens 1959). Such a system might exist in certain species or

77 become operative under certain conditions. It is therefore worthwhile to look out for a quantum requirement of less than 8 per oxygen molecule. Because the attainment of such a low requirement appears unlikely at the moment, a method of measurement should be developed, avoiding uncertainties in assumptions and conditions of measurements, and preferably easily duplicated by other investigators. A low quantum requirement would prove that under these conditions at least part of the electron transport would not occur via two photochemical one-quantum reactions in series. Another question is whether the work reported in my thesis could have been done earlier. I think it could have been done about ten years earlier. Similar photoelectric cells as I used were then available, but perhaps not the sensitive amplifier, which was needed for measuring the fluorescence spectra. This amplifier, however, could have been compensated by a more powerful monochromator than I had at my disposal. Also available in 1937 at the Utrecht physical laboratory were the electronic components needed for constructing a simple but sensitive absorption difference spectrophotometer. No theory was available for the calculation of the transfer of electronic excitation energy, and thus for estimating the maximum number of pigment molecules per reaction center, which would have given some indication of the required spectrometer sensitivity. But if nevertheless a spectrophotometer had been constructed and correctly applied to various groups of algae and purple bacteria, the discovery of cytochrome oxidation and reaction centers in photosynthesis would have taken place perhaps five years earlier, not ten years, because of the war, the smaller number of scientists involved, and the nonexistence of equipment such as photomultipliers and recorders. A more difficult question t o answer is what would have happened if I had not introduced absorption difference spectroscopy into photosynthesis in 1952. Without taking into account other developments, I think that it would have been introduced by other people possibly within 2, and probably within 15 years. Other developments occurred, however, in photosynthetic research from 1952 :onwards, which were independent of absorption ]difference spectroscopy, and make a more precise estimate possible: the discovery of photophosl~horylation (Arnon et al. 1954, 1958)

and of the occurrence of cytochromes in chloroplasts (Davenport and Hill 1952) and photosynthetic bacteria (Vernon 1953). Analogy with respiratory phosphorylation would have suggested a connection between cytochrome, photophosphorylation and photosynthesis and would probably have led to an attempt to demonstrate and study light induced cytochrome redox reactions, which would have stimulated the construction of a photosynthetic absorption difference spectrophotometer. Thus the delay of this application would have been probably less than 4 years. This illustrates the potential power of the attack on a scientific problem from many directions.

3.2. The discovery of the two photosynthetic systems in oxygenic photosynthesis was bound to happen in the early sixties As discussed, my measurements of action spectra and kinetics of cytochrome oxidation in Porphyridium cruentum, which provided convincing evidence in favor of the two reaction center hypothesis, were started in order to check or rather refute certain conclusions from Emerson's enhancement experiments. The interpretation of these measurements was facilitated by the knowledge concerning the existence of two kinds of chlorophyll a with different functions. I think, however, that after the introduction of absorption difference spectroscopy and the discovery of the photooxidation of reaction centers and cytochromes in purple bacteria, followed by that of cytochromes and P700 in algae, the discovery of the two photosynthetic systems was bound to happen in the sixties, independent of previous discoveries or speculations. Because of its clear cytochrome f spectrum, Porphyridium would probably have been selected for kinetic and spectroscopic studies and the completely different kinetics, obtained when illuminating with green light instead of red, for instance for measuring an action spectrum for cytochrome oxidation, would have revealed the presence of a second photochemical system (see Duysens 1960). Without Emerson's, Kok's or my experiments, other research groups would probably have carried out these experiments at a not much later date. Generalizing, I suggest, that as soon as a new

78 powerful method or theory has been developed in an active field of science, the subsequent discoveries based on the application of this method or theory will presumably only marginally depend on the individual investigators, although the prizes usually will go to the best scientists and/or to the richest in manpower and resources. If the top ten were absent, another top ten would take its place, and the evolution of the field may be somewhat slower. The introduction of new methods and theories will be enhanced in a field in which a great variety of experimental approaches is already being used.

Acknowledgements I am indebted to Drs Govindjee, Jan Amesz and Hans van Gorkom for valuable suggestions concerning the manuscript.

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