PhotosynthesisResearch 43: 177-189, 1995. © 1995KluwerAcademicPublishers. Printedin the Netherlands. Personal perspective

The discovery and function of plastocyanin: A personal account* Sakae Katoh Department of Biology, Faculty of Science, Toho University, Miyama, Funabashi 274, Japan Received 1 January 1995;accepted23 January1995

Key words: algal cytochrome c553, Hiroshi Tamiya, Keita Shibata, photosynthetic electron transport, plastocyanin, Photosystem I

Abstract A brief autobiographical account is presented of the early research that led to the discovery of the copper protein plastocyanin and the identification of its function as an electron carrier in plant photosynthesis. A discussion follows of different approaches employed for the determination of the functional site of plastocyanin in relation to cytochromef. A surranary is provided of a heated controversy about the involvement of two or three light reactions in photosynthesis and an experiment is described that has contributed to resolution of the controversy through the identification of the functional site of plastocyanin. An early history of photosynthesis research in Japan is also discussed.

Abbreviation: DCIP - 2,6-dichlorophenolindophenol Towards research into photosynthesis When I entered the University of Tokyo in 1951, it was my intention to focus my future studies within agricultural sciences. The reason was very simple. I was born in 1932 in Kobe, the largest port in western Japan which has very recently been severely damaged by earthquake, but my family moved to the countryside to avoid the anticipated air raids near the end of the World War II. The area in which we lived was rich farmland with numerous rice paddies. However, we suffered from a considerable shortage of food since agricultural production had fallen catastrophically during the war. It seemed to me at that time that there was nothing more important than improving food production. The more I learned at the university, however, the more I was attracted by the basic biological sciences. Plants became familiar to me through several trips to mountain areas for the collection of plant specimens in my first and second year at the university. The food * Invitedand editedby Govindjee.

situation had improved by that time. In 1955, I joined Prof. Hiroshi Tamiya's laboratory at the Institute of Botany to begin my graduate studies in plant physiology. I did not see Prof. Tamiya often in the Institute of Botany because he remained at the Tokugawa Institute most of the time (see below) and I was allowed to do anything I wanted. The staff members of the laboratory were Hirosi Huzisige (University of Okayama) and Shigehiro Morita (Tokyo University of Agriculture and Technology). Morita taught me how to start experiments. Among the graduate students were Seikichi Izawa (Wayne State University), Shigetoh Miyachi (University of Tokyo) and Mitsuo Nishimura (University of Kyushu). I learned a lot by watching how they designed and executed their mostly successful but occasionally fruitless experiments. As suggested by Morita, I started my research by examining the effects of light on the metabolism of ~4C-labeled lactate in the purple bacterium Rhodopseudomonas palustris. Scientific activities at the university had been essentially non-existent for some time after the war and, although the most difficult time was over, the

180 underground laboratory in which I worked was nothing but a deserted cellar with junk scattered all around. The first thing I did was to find a motor and other bits and pieces for the construction of a temperaturecontrolled shaking incubator. My experience in this poorly equipped laboratory turned out to be very useful when I started my own laboratory with empty rooms and a meager budget in the Department of Pure and Applied Sciences in 1973. Kazuhiko Satoh joined me and we constructed a flash spectrophotometer and other apparatus by ourselves. Our efforts, together with the two unique organisms that we employed, namely, the thermophilic cyanobacterium Synechococcus elongatus, which has very stable proteins (Yamaoka et al. 1978), and the green alga Bryopsis maxima, which provided us very stable intact chloroplasts (Katoh et al. 1975), enabled us to perform a series of important experiments at extremely low cost. I spent more than a year working with the purple bacterium without any exciting results. Intact cells of the bacterium seemed to me to be too complicated a network of metabolic pathways for analysis. I decided, therefore, to work with a simpler, cell-free system and soon I found that light suppressed respiratory uptake of oxygen in membrane fractions (chromatophores) as strongly as in cells. Because the effect of light was unaffected by the addition of ADP or the uncouplers, I concluded that a competition must exist between oxygen and an oxidant produced by light for respiratory substrates. This study resulted in my first paper (Katoh 1961a), although its publication was delayed for some reason I cannot now recall. However, I have a clear memory of Prof. Tamiya, who, after reading my manuscript, said to me 'kabe dane', which means 'you are at an impasse'. To myself, I said silently, 'Oh, no, I am not, sir', since I had several plans, but did not dare to voice my opinion to my eminent professor. I continued to work with chromatophores for some time, but Tamiya's words haunted me. Eventually, I gave up the project and started an investigation into cytochromes in algae, which led me to the discovery of plastocyanin.

The early history of photosynthesis research in Japan Govindjee suggested to me that I should describe how investigations into photosynthesis were initiated and developed in Japan in this article since this topic is not familiar to scientists elsewhere. It is not possi-

Fig. 1. Prof.Keita Shibata (1877-1944). The photograph was taken in 1935, two years after the publicationof the paperon the inhibitoryeffectsof hydroxylamineon photosynthesisby Yakushiji and himself.Courtesyofthe late Prof.KozoHayashi,his son-in-law. ble to outline the entire history of such research in the present limited perspective (see Huzisige and Ke 1993 for a perspective on the history of photosynthesis research). I shall briefly describe the history of our laboratory at the University of Tokyo because this history corresponds essentially with the early history of photosynthesis research in Japan. The first paper on the mechanism of photosynthesis by Japanese authors had appeared in 1933. The authors of the paper, entitled 'Der Reactions Mechanismus der Photosynthese', were Prof. Keita Shibata (Fig. 1, also see Tamiya 1955) of the Institute of Botany at the Imperial University of Tokyo (University of Tokyo after the war) and his student Eijiro Yakushiji (Toho University). In their paper, they showed for the first time that hydroxylamine is an inhibitor of photosynthesis. Because hydroxylamine was known to inhibit catalase, this finding led them to the conclusion that all oxygen molecules are produced from H20 via H202

181 in photosynthesis. They proposed that the light reaction of photosynthesis is photoactivation (photolysis) of 4H20 molecules to 4[H--OH], then 4[tHis form 02 and 2H20, while 4[His are utilized for the reduction of CO2 to formaldehyde. (We, of course, know now that formaldehyde is not the product but the paper was published about 20 years before the discovery of the Calvin-Benson cycle). Shibata had already elaborated his idea of photoactivation of water in his monograph 'Carbon and Nitrogen Assimilation' published two years earlier (Shibata 1931). There he extended his idea to bacterial photosynthesis: after activation of H20, [HI is transferred to CO2, while [OH] is used as oxidant for electron donors. This idea is very similar to the generalized hypothesis about bacterial and plant photosynthesis proposed by Van Niel (see e.g., Van Niel 1941). Unfortunately, Shibata's monograph was written in Japanese and was unavailable to Western scientists until 1975, when the text was translated into English by Howard Gest and Robert K. Togasaki. For a complete history of these concepts, the reader should also consult papers by Thunberg (1923), Wurmser (1930), Van Niel (1930), Van Niel and Muller (1931) and Gest (1993). Four of Shibata's students, namely, Hiroshi Tamiya, Eijiro Yakushiji, Hiroshi Nakamura and Atusi Takamiya inherited Shibata's interest in photosynthesis. Yakushiji found cytochromes in higher plants and algae and extracted a cytochrome from a red alga (Yakushiji 1935). This was the first electron carder of photosynthesis isolated, although no one envisioned its function in photosynthesis at that time. Yakushiji was also one of the discoverers of a water-soluble chlorophyll protein in Chenopodium leaves (Yakushiji et al. 1963). Nakamura studied photosynthesis of purple bacteria and observed the production of H2 from certain bacteria (Nakamura 1937). Hiroshi Tamiya (Fig. 2, also see Takamiya 1963) succeeded Prof. Shibata at the Institute of Botany in 1943. He performed detailed quantitative analyses of the mechanism of photosynthesis both using inhibitors (Tamiya and Huzisige 1942) and intermittent light (Tamiya and Chiba 1949a,b). When 14C became available in Japan, he and Shigetoh Miyachi undertook a series of kinetic investigations of different steps in photosynthesis by measuring fixation of CO2 in preilluminated Chlorella cells (Tamiya et al. 1957). Tamiya also served as the director of the Tokugawa Institute for Biological Research and supervised two series of important investigations into the growth of Chlorella there. First, introduction of new methodolo-

Fig. 2. Prof.HiroshiTamiya(1903-1984). Drawnby a streetartist in Istanbulin 1950.Courtesyof Prof.EijiHase.

gy, namely, synchronization of growth phases, allowed the cellular life-cycle of the alga to be analyzed in detail (Tamiya et al. 1953; Tamiya 1963). His second contribution was development of the methods for mass culture of the unicellular alga. He established a method of outdoor culture suitable for a commercially viable enterprise (Tamiya 1957). Tamiya's scientific achievements and warm hospitality attracted many young scientists from different disciplines. Eiji Hase, a chemist, joined the group after the war and later succeeded Tamiya at the Research Institute of Applied Microbiology. During the war, Tamiya was asked to investigate the medical effects of certain dyes by a research branch of the Japanese army. During this project, a young lieutenant by the name of Kazuo Shibata made the acquaintance of Tamiya and became a member of the Tokugawa Institute after Japan was disarmed. One day, Shibata (not to be confused with Keita Shibata) was asked to record absorption spectra of suspensions of algal cells. This task led him to develop the opal glass method, which allows absorp-

182 tion spectra of turbid suspensions to be recorded with minimum interference from light scattering (Shibata et al. 1955). Later he organized, together with Yorinao Inoue and Teruo Ogawa, an active research group in the Institute of Physical and Chemical Research (RIKEN), Wako. Tamiya moved to the newly founded Research Institute for Applied Microbiology at the University of Tokyo in 1955, and his position at the Institute of Botany was taken over by Atusi Takamiya. When the Department of Biophysics and Biochemistry was established in 1959, Takamiya's group joined the new Department. I would like to emphasize that the photosynthesis community in Japan is no longer as dynastic as used to be. I am pleased to note that a large number of scientists with different scientific backgrounds are currently engaged in research in photosynthesis. However, researchers who are related directly or indirectly to the Shibata-Tamiya-Takamiya's school still account for the largest proportion of the community, l

Studies on algal cytochrome c-553 Kinetic analysis was a favorite tool for studies of photosynthesis and other biological reactions in Tamiya's laboratory. I followed this tradition in my studies with the purple bacterium, but I began to feel after a while that this approach left a large 'black box' untouched unless more were known about the biochemistry of the reactions. In the 1950's, evidence began to accumulate suggesting that cytochromes are involved in photosynthesis. Cytochromesfand b6 were found in chloroplasts by Hill and coworkers (Hill and Scarisbrick 1951), and their findings were followed by the discovery of Vernon and Kamen (1954) that photosynthetic bacteria contain large amounts of c-type cytochromes. i The followingpeoplefrom Takamiya'sgroup are working in the fieldof photosynthesisand in relatedsubjectsat the timeof this writing: TetsuyaKatoh (Universityof Kyoto),Ryuji Kanai (University of Saitama), HidehiroSakurai (WasedaUniversity),Norio Murata (NationalInstitutefor BasicBiology),Ken-ichiroTakamiya (TokyoInstituteof Technology),KazuhikoSatoh (I-IimejiInstitute of Technology),MitsumasaOkada(TohoUniversity),IsamuIkegami (TeikyoUniversity),ShigeruItoh (National Institute for Basic Biology)and myself. It should also be notedthat KimiyukiSatoh is from Prof. Huzisige's laboratoryat the Universityof Okayama and Prof. KazuoOkunuki, a student of Prof. Keita Shibata, fostered the talents of pupils such as TakekazuHorio (Universityof Osaka), MasateruShin(Universityof Kobe)and TakashiYamashita (Universityof Tsukuba).

Although photooxidation of cytochrome f (or a similar pigment) in algal cells had been reported by Lundeggtrdh (1954) and Duysens (1955), less was know about biochemical properties of cytochromes in algae. I started to work with a cytochrome from the red alga Porphyra tenera in 1958. Several people said to me, 'You'd better use a material unique to Japan', and others said more explicitly, 'Don't compete with scientists abroad. You'll never win'. These statements might sound ridiculous now but we were very heavily handicapped by our poor research conditions at that time. Porphyra tenera seemed to me to be an appropriate experimental material since it is a unique and favorite food (nori) in Japan that was at that time cultivated on the sea coast of Tokyo. I assumed that I should be able to work with this organism without any competition. This assumption turned out to be incorrect, however, and soon I was involved in a competition with two groups in Japan (see below). After I had isolated and purified a c-type cytochrome from the alga, I learned that the cytochrome had already been reported by Yakushiji as early as 1935, although he considered the cytochrome with an asymmetric a-band to be a complex of two cytochromes (Yakushiji 1935). I found that cytochrome c-553 was located in the thylakoids and had a redox potential that was higher than that of cytochrome c in the mitochondrion, but similar to that of cytochrome f in the chloroplast (Katoh 1959a, 1960b). The reduced cytochrome was oxidized by the thylakoid fraction in the light but not in the dark (Katoh 1959b). A similar cytochrome was found in a wide variety of algae (Katoh 1959a). I concluded, therefore, that cytochrome c-553 should play a role in photosynthesis but not in respiration. In the early sixties, purification of proteins was not a simple task and crystallization of a protein was a success worthy of prompt publication in a journal like Nature. So I proceeded to crystallize the cytochrome. It happened that Prof. Okunuki's group at University of Osaka and one more group in Japan had started the same project. Okunuki's group had already succeeded in the crystallization of several cytochromes using column chromatography with a cation-exchange resin for purification. Because Porphyra cytochrome c-553 is an anionic protein, DEAE-cellulose, an adsorbent with anion-exchange properties which had been synthesized by Peterson and Sober (1956), seemed to me more promising. Because no commercial product was yet available, I synthesized DEAE-cellulose by myself and spent several days in a hospital as a result of my

183 ignorance of the harmful effects on my eyes of one of the chemicals that I used. The adsorbent turned out to be an excellent tool for purification of the cytochrome and I was able to crystallize the cytochrome in a short period of time (Katoh 1960a). Okunuki's group also succeeded in crystallization of the cytochrome at about the same time, and the third group did much later.

Discovery of plastocyanin In the year of 1959, I was given about one kilogram of freeze-dried cells of Chlorella ellipsoidea, the product of mass culture of the alga at the Tokugawa Institute. Unexpectedly, I was unable to find cytochrome c-553 in repeated attempts with small amounts of the sample. Therefore, I decided to use the whole batch of the cells that remained for extraction of cytochrome c-553. When the extract was passed over a large column of DEAE-cellulose, a number of fractions ranging from pale yellow to red in color were separated. No cytochrome c-553 could be detected. However, one of the yellow fractions was an NADPH-specific flavoprotein (Katoh 1961b), and a red fraction was later found to contain ferredoxin. During rechromatography of a yellow fraction, I noticed that a green band appeared and then gradually faded into the yellow background. I was curious and repeated the column chromatography of the fraction. The green color reappeared temporarily in some cases but not in others. Eventually, I found that ferricyanide was effective in keeping the substance in question green and this discovery greatly facilitated purification of the substance. The purified substance was blue in color, resembling laccase, a copper-enzyme, which happened to be under investigation in a neighboring laboratory. The blue substance was indeed a copper-containing protein (Katoh 1960c). As expected from its behavior during column chromatography, the protein had no affinity for oxygen, but was oxidized by ferricyanide and reduced by various reductants. Looking into the literature, I found two lines of evidence to suggest that copper might be involved in photosynthesis. (1) A major portion of the copper in green leaves is located in chloroplasts (Neish 1939; Whatley et al. 1951), and (2) several copper-specific inhibitors suppress photosynthesis more strongly than respiration in Chlorella cells (Greene et al. 1939). These observations impelled me to search for a function of the copper protein in photosynthesis. We found that the protein was also present in green leaves of vari-

ous higher plants, but neither in non-chlorophyllous tissues, such as roots of carrot and turnip, nor in photosynthetic bacteria (Katoh and Takamiya 1961; Katoh et al. 1961). In leaves, the protein was located in chloroplasts. These data convinced me that we were on the right track. In view of its localization in chloroplasts and characteristic blue color of the oxidized form, the protein was named plastocyanin (Katoh and Takamiya 1961). To my disappointment, none of the inhibitors of copper enzymes that we examined seemed to affect or to interact with the copper of plastocyanin (Katoh and Takamiya 1964). Only harsh treatments, such as incubation at pH 2.0, or dialysis against 10 mM KCN at pH 8.0 for 24 h, were effective in removing copper from the protein. These treatments seemed to me too drastic for application to chloroplasts or algal cells [treatment with KCN was, however, employed for inactivation of plastocyanin in situ by Izawa et al. (1973)]. We also examined the effects of Hg 2+ because involvement of a sulfhydryl group in the binding of copper to hemocyanin was being discussed at that time. Hg 2+ induced only very slow bleaching of the blue color of the oxidized protein unless the protein structure had been loosened by treatment with a high concentration of urea (Katoh and Takamiya 1964). We concluded, therefore, that Hg 2+ was not a good inhibitor of plastocyanin. As will be described below, however, this premature conclusion resulted in a long delay in the elucidation of the function of the protein. I had little opportunity to discuss the function of plastocyanin in photosynthesis with scientists abroad because we were very much isolated from the photosynthetic community in the Western countries. Shortly after my discovery of plastocyanin, Martin Kamen visited us and, after a very one-sided discussion, he suggested that I might write to Robin Hill for his opinion about the copper protein, and indeed I did. His answer was not, however, very encouraging. He wrote that, after reading my article in Nature, he had also found plastocyanin in the plants he was studying. His letter ended as follows, 'There are several blue Cu-proteins that have been described from different animal tissues but here there is no evidence that they are related to the cytochrome system except that cytochrome oxidase preparations have been found to contain Cu'. Several people also suggested to me that plastocyanin might be one of the blue copper proteins with unknown functions, or with no function at all. Nonetheless, I persisted in my work with plastocyanin. I reasoned that since plastocyanin has a

184 redox potential of 370 mV, only 5 mV above that of cytochrome f, the functional site of the copper protein should be close to cytochrome f or photosystem I (PS I). The following observations supported my hypothesis. Plastocyanin was photoreduced by thylakoids but, when the photoreducing activity was suppressed by treatment of the membranes with detergents, the reduced protein was rapidly oxidized in the light (Katoh and Takamiya 1963b). Plastocyanin also greatly accelerated photooxidation of cytochrome c by detergent-treated thylakoids (Katoh and Takamiya 1963b). Kok et al. (1964) showed that plastocyaninenhanced photooxidation of cytochrome c is most efficiently sensitized by long wavelength light. Thus, plastocyanin is oxidized by PS I. Earlier work had revealed a soluble and heat-labile factor in chloroplasts that stimulated photoreduction of indigo carmin with reduced DCIP (Vernon and Hobbs 1957) or photooxidation of cytochrome c (Nieman and Vennesland 1959). Plastocyanin also accelerated photoreduction of indigo carmine (Katoh and Takamiya 1963b, 1965a). The soluble factor and plastocyanin had the same pattern of distribution and molecular properties. Only the molecular size of the factor appeared to be significantly smaller than that of plastocyanin, but our determination of the size of the plastocyanin molecule turned out to be an overestimate. Thus, the factor and plastocyanin were one and the same protein. Several groups of investigators suggested that plastocyanin is reduced by PS II and hence serves as an electron carrier between P S I and PS II (Katoh and Takamiya 1963a; Trebst and Eck 1963; Kok et al. 1964; de Kouchkovsky and Fork 1964). Evidence presented was, however, not necessarily convincing. We observed photoreduction of plastocyanin by thylakoids (Katoh and Takamiya 1961, 1963b) but the protein is most likely reduced by P S I rather than PS II in this reaction. Trebst and Eck (1963) suggested the participation of plastocyanin in photosynthetic electron transport because salicylaldoxime, an inhibitor of copper enzymes, strongly suppressed NADP + photoreduction with water as electron donor. We showed later, however, that salicylaldoxime inhibits PS II electron transport (Katoh 1972) but not plastocyanin (Katoh and San Pietro 1966b). De Kouchkovsky and Fork (1964) found a light-induced absorbance increase at 591 nm in algal cells and isolated chloroplasts and attributed it to the oxidation of plastocyanin. They proposed that plastocyanin functions between the two photosystems based upon the observation that PSI and PS II lights had antagonistic effects on the 591 nm

response. I doubt, however, that the 591 nm response is related to plastocyanin because the magnitude of the 591 nm absorbance increase, which corresponds to about half that of the 518 nm electrochromic band shift (de Kouchkovsky and Fork 1964), is at least one order of magnitude larger than would be expected from the oxidation of all the plastocyanin molecules present in chloroplasts. Generally, it is difficult to measure accurately absorbance changes of plastocyanin in situ because of overlapping absorption changes as well as its small absorption coefficient. I became confident in that plastocyanin is an intrinsic electron carrier functioning between the two photosystems when we found that inactivation of photoreduction of the physiological electron acceptor NADP + is well correlated with release of the copper protein (Katoh and Takamiya 1965b; Katoh and San Pietro 1966a). Disruption of thylakoid membranes by sonic oscillation resulted in a strong inhibition of NADP + photoreduction, either water or reduced DCIP as electron donor, concomitant with the release of plastocyanin, while PS II-mediated photoreduction of ferricyanide or DCIP was resistant to the treatment. This shows that sonic treatment blocks electron transport at or near PS I. The treated membranes were also active in photoreduction of plastocyanin but, when PS II electron transport was blocked by an inhibitor, the reduced plastocyanin was photooxidized by the preparation. Furthermore, the lost activity of NADP + photoreduction with water as electron donor was effectively restored by the addition of plastocyanin to sonicated membranes. We concluded, therefore, that plastocyanin is an intersystem electron carrier that functions near PS I. One day, I received a telephone call from a man who wanted to know whether I could speak English. I was not available but Nishimura responded, 'I suppose he can.' So I was invited to the Symposium on Photosynthetic Mechanisms of Green Plants, organized by Bessel Kok and Andr6 Jagendorf at the Airlie House, Virginia, in 1963 and presented a paper on plastocyanin (Katoh and Takamiya 1963a). This was my first trip abroad and I met many distinguished people whom I had previously known only from the literature. I was greatly impressed by their warm attitude to a young scientist from the Far East. I stayed in the C. F. Kettering Laboratory, in Yellow Springs, Ohio, from October 1964 to December 1966, working with Tony San Pietro. One of our results surprised me: there was no plastocyanin in the cells of Euglena gracilis and cytochrome c-553 connected PS

185 I and PS II in the place of plastocyanin (Katoh and San Pietro 1967). I was confused because at that time I believed cytochrome c-553 to be cytochromef It was only much later that cytochrome c-553 was shown to correspond to plastocyanin, and not to cytochrome f (Wood 1977, 1978; Bohner and BOger 1978). In contrast to higher plants, which contain only plastocyanin, algae contain either plastocyanin or cytochrome c-553, or both. Thus, Chlorella ellipsoidea has only plastocyanin, but Euglena gracilis lacks the copper protein.

Functional site of plastocyanin In the decade that followed the discovery of plastocyanin, several different functional sites of plastocyanin in relation to cytochromefwere proposed. P-700

. , I plastocyanin

(1)

""- cytochrome f

P-700 ~-- cytochrome f -.-- plastocyanin

(2)

P-700 ~

cytochrome f

(3)

PS IIb

(4)

plastocyanin ~

PS IIa -*-- plastocyanin ~ P-700 ~

cytochrome f ~-]

[

The notion that plastocyanin and cytochrome f donate electrons to P-700 in parallel (sequence (1)) was put forward on the basis of the observations that both are photooxidized at similar rates in detergent-treated thylakoids (Kok et al. 1964). As mentioned above, however, Euglena cytochrome c-553 employed in this study was not cytochrome f. In sequence (2), plastocyanin was placed before cytochromefbecause salicylaldoxime abolished the 591 nm absorption change and the photoreduction of cytochrome f (Fork and Urbach 1965). However, the significance of this observation is not clear for the reasons discussed above. Sequence (3) was supported by, among others, the observation that a plastocyanin-deficient mutant of Chlamydomonas reinhardtii was inactive in P S I activities, such as photooxidation of cytochrome f (membrane-bound cytochrome c-553) or photoreduction of NADP + with reduced DCIP (Gorman and Levine 1966). Addition of plastocyanin restored the P S I activities. By contrast, a mutant strain deficient in cytochrome f was able to photoreduce NADP + in the absence of added plastocyanin. I considered that

the data from the algal mutants were quite convincing and, therefore, I was very surprised when the late Professor Daniel Arnon's group reported results that cast a strong doubt on the role ofplastocyanin as an electron donor to P-700. Michel and Michel-Wolwertz (1969) separated PS I and PS II particles, which corresponded to the stroma thylakoids and grana stacks, respectively, from thylakoid membranes that had been disrupted by passage through a French pressure cell. Arnon et al. (1970) claimed that this PSI preparation mostly lacks plastocyanin but was able to reduce NADP + with reduced DCIP at a high rate, with added plastocyanin having only a marginal effect. This result was taken as evidence for their hypothesis of three light reactions in photosynthesis (Knaff and Arnon 1969; Arnon et al. 1970). They proposed that P-700 accepts electron from cytochrome f in cyclic electron transport, while plastocyanin functions in linear electron transport from water to NADP + which involves two PS II centers (sequence (4)). They argued that NADP + photoreduction with reduced DCIP is mediated by P S I (P-700) and, hence, proceeds in the absence of plastocyanin. The new model invited heated arguments among investigators because it challenged the widely held concept of only two light reactions in photosynthesis. The functional site of plastocyanin suddenly became the focus of attention since it provided a critical and feasible test of the involvement of two or three light reactions in photosynthesis. The results of experiments by other groups of investigators added to the confusion. Consistent with the observation of Arnon's group, Fork and Murata (1971a,b) reported that the P S I particles contained only a negligible amount of plastocyanin but could oxidize cytochrome f rapidly in the light. They claimed that electron transport from cytochromefto P-700 does not involve plastocyanin and that the stimulation of PS I electron transport by added plastocyanin is an artifact introduced by disruption of membrane structures. However, other groups found a substantial amount of plastocyanin in P S I preparations (Baszynski et al. 1971; Arntzen et al. 1971; Sane and Hauska 1972). The apparent lack of a requirement for plastocyanin in the photoreduction of NADP + or the photooxidation of cytochrome f was, therefore, ascribed to the presence of the bound plastocyanin in the PSI preparation. Investigations with antisera raised against the copper protein also yielded equivocal results as to the functional site of plastocyanin. The function of the copper protein was a main topic of a symposium that was orga-

186 nized by the late Professor Gtinter Jacobi at G~Sttingen in 1971. No consensus was reached after three days of heated discussion because of the largely contradictory data available at that time. The controversy was finally settled by an experiment in which we originally intended to identify an inhibitor of PS II electron transport or oxygen evolution. I suggested to Mamiko Kimimura, a graduate student, that she examine the effects of Hg 2+ to determine whether sulfhydryl groups are involved in oxygen evolution. Her results were very surprising to me. Treatment of thylakoids with Hg 2+ for several minutes had no effect on PS II electron transport but strongly inhibited a P S I reaction, namely, photoreduction of methyl viologen with reduced DCIP as electron donor (Kimimura and Katoh 1972). Hg 2+treatment also resulted in the inhibition of the photooxidation of cytochrome f, but not that of P-700. Thus, it appeared that Hg 2+ specifically blocked electron transport between cytochrome f and P-700, a site at which plastocyanin is considered to function in the two light reaction model of photosynthesis. I was excited but at the same time puzzled by this finding because early studies had shown that Hg 2+ is a poor inhibitor of plastocyanin (Katoh and Takamiya 1964). Soon it occurred to me that we had investigated the effects of Hg 2+ only on the oxidized plastocyanin since we had used its blue color as a convenient indicator of the copper bound to the protein. If plastocyanin were present in the reduced state in chloroplasts and the reduced protein were more susceptible to Hg 2+ than the oxidized protein, the observed effect of Hg 2+ could be ascribed to the inactivation of the plastocyanin. Such was indeed the case. In contrast to the results with the oxidized protein, which is strongly resistant to Hg 2+, only a brief incubation was needed for the total replacement of copper in the reduced protein by Hg 2+. The Hg2+-inactivated chloroplasts contained only Hg 2+substituted plastocyanin. Furthermore, inhibition was greatly weakened if the thylakoids were treated with ferricyanide to oxidize the protein prior to treatment with Hg 2+. It was concluded, therefore, that Hg 2+ blocks electron transport by specifically attacking plastocyanin. Thus, the functional site of plastocyanin was finally established to be between cytochrome f a n d P700. It took twelve years for me to identify plastocyanin as the primary electron donor of P S I since my discovery of the copper protein in 1960. Our premature conclusion that plastocyanin is not very sensitive to Hg 2+ (Katoh and Takamiya 1964) resulted in a long

delay in the elucidation of the function of plastocyanin. However, our findings were made just in time to settle the controversy as to whether photosynthesis involves two or three light reactions. Our data were consistent with the two light reaction model and ruled out Arnon's three light reaction model, which assumes that P700 receives electrons directly from cytochromefand plastocyanin functions in a separate electron transport chain (sequence (4)). Some time later, David Knaff, who had been actively engaged in investigations related to the three light reaction hypothesis, told me that after reading our paper he gave up the hypothesis. Various studies that followed our experiment also provided evidence against sequence (4). The three light reaction model was consigned to the long history of photosynthetic research. Finally, I shall briefly describe the recent advances in research into the function of plastocyanin (for reviews, see Sykes 1991 and Gross 1993). Subunit III of P S I reaction center complex, the product of the psaF gene, was implicated as the plastocyaninbinding protein (Hippler et al. 1989, but see Chitnis et al. 1991). The interaction of plastocyanin with its reaction partners, cytochrome f and P-700, is electrostatic in nature and strongly influenced by ionic strength and pH of media. Because plastocyanin from higher plants is a negatively charged molecule, its binding to positive charges on the partner molecules was suggested (Gross 1993). The crystal structure of poplar plastocyanin, which was determined by Colman et al. (1978), has greatly contributed to elucidation of the mechanism of electron transfer between plastocyanin and its reaction partners. The copper atom, which is ligated to a thiol group of Cys 84, a thioester group of Met 92 and two imidazol groups of His 37 and His 87, is embedded in the protein molecule. Electron transfer to and from the copper center, therefore, proceeds through amino acid residues. There are two potential sites on the protein molecule, which are considered to participate in the interaction with the reaction partners. Different approaches, in particular, chemical modification and site-directed mutagenesis showed that Tyr 83 and the surrounding patches of negative charges are involved in the binding of plastocyanin to cytochrome f. Another site is His 87, which separates the copper atom from the medium. Electrons are suggested to be transferred from copper via an outer-sphere mechanism through the coordinated imidazole group of this residue (Coleman et al. 1978). Recently, Martinez et al. (1994) have crystallized and obtained the structure of the hydrophilic portion of cytochrome f

187 from turnip chloroplasts. The three dimentional structure of P S I reaction center complex from the thermophilic cyanobacterium Synechococcus elongatus is under investigation (Krauss et al. 1993). I expect that our understanding of the mechanism of electron transport in the plastocyanin region will be greatly advanced in the near future.

numerous investigators. I feel that I have been very fortunate to have been able to witness the amazing development of our understanding of photosynthesis during this exciting period of time. I have really enjoyed these 40 years.

Acknowledgement Epilogue Looking back and reviewing my own research is not really as important and exciting to me as what I am going to do next. However, when I retired from the University of Tokyo in 1993, I had to review my career as a scientist because a retiring professor is expected to give an overview of his achievements in his last lecture of the university. Preparing for the lecture, I found that I could recall many experiments that I had done 30 years ago with greater clarity than the more recent ones, perhaps because as a beginning scientist everything I did, not only my successes but also my failures, was so new and exciting to me. Since the audience of m y final lecture consisted mostly of young people, I spent the first half of my lecture talking about my early research. What I have written here is mostly based upon this part of my lecture. The occasion gave me a chance to count all my past collaborators: I was surprised to find that I have had more than seventy coauthors on my papers. They are or were mostly young and able investigators. I would like to take this opportunity to express my hearty appreciation to all of them for our joyful and inspirational collaboration. I have also been impressed by the tremendous changes that have occurred in the environment that surrounds science in Japan since I started my first experiment in an ill-equipped underground laboratory. I have touched briefly on the difficult situation that we found during the post-war period, thinking that it might be interesting to young scientists, in particular, to those working with insufficient facilities in developing countries. Research into photosynthesis has advanced with ever-increasing speed since I joined the Tamiya's group in 1955. Major discoveries have been followed by more major discoveries and the mechanism of this highly sophisticated and efficient system of plants has been elucidated to an extent that no one could have imagined forty years ago. To me, photosynthesis research in the past four decades has been a fascinating drama full of new ideas and discoveries, interwoven with successes and failures, and joy and disappointments of

I thank Govindjee for carefully editing this perspective.

References Arnon DI, Chain RK, McSwaln BD, Tsujimoto HY and Knaff DB (1970) Evidence from chloroplast fragments for three photosynthetic light reactions. Proc Natl Acad Sci USA 67:1404-1409 ArntzenCJ, DilleyRA, Peters GA and ShawER (1971) Photochemical activity and structural studies of photosystems derived from chloroplast grana and stroma lamellae. Biochim Biophys Acta 256:85-107 Baszynski T, Brand J, KrogmarmDW and Crane FL (197t) Plastocyanin participation in chloroplast PhotosystemI. Biochim Biophys Acta 234:537-540 Bohner H and B0ger P (1978) Reciprocalformation of cytochrome c-553 and plastocyaninin Scenedesmus. FEBS Lett 85:337-339 Chitnis PR, Purvis D and Nelson N (1991) Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of Photosystem I from the cyanobacteriumSynechocystis sp. PCC 6803. J Biol Chem 266:20146-20151 ColmanPM,FreemanHC, Guss JM, Murata N, Norris VA,Ramshaw JAM and VenkatappaMP (1978) X-ray crystal structure analysis of plastocyanin at 2.7 ,~ resolution. Nature (London) 277:319324 de KouchkovskyY and Fork DC (1964) A possible functioningin vivo of plastocyanin in photosynthesis as revealed by a lightinduced absorbance change. Proc Natl Acad Sci USA 52: 232239 Duysens LNM (1955) Role of cytochromeand pyridine nucleotide in algal photosynthesis. Science 121:61-79 Fork DC and Murata N (1971a) Oxidation-reduction reactions of P700 and cytochromef in fraction 1 particles prepared from spinach chloroplasts by French press treatment. PhotochemPhotobiol 13:33-44 Fork DC and Murata N (1971b) Photochemically-activeparticles from chloroplasts fragmentedin a French pressure cell. In: Forti G, AvronM and Melandri A (eds) Proc 2nd Intl CongrPhotosynth Res, Vol I, pp 847-857. Dr. Junk Publishers, The Hague Fork DC and Urbach W (1965) Evidence for the localization of plastocyanin in the electron-transport chain of photosynthesis. Proc Natl Acad Sci USA 53:1307-1315 Gest (1993) History of concepts of the comparative biochemistry of oxygenicand anoxygenicphotosynthesis. PhotosynthRes 35: 87-96 GormanDS and LevineRP (1966) Photosyntheticelectrontransport chain of Chlamydomonas reinhardi VI. Electron transport in mutant strains lacking either cytochromec-553 or plastocyanin. Plant Physio141:1648-1656

188 Greene LF, McCarthy JF and King CG (1939) Inhibition of respiration and photosynthesis in Chlorella pyrenoidosa by organic compounds that inhibit copper catalysts. J Biol Chem 128: 447453 Gross EL (1993) Plastucyanin: Structure and function. Photosynth Res 37:103-116 Hill R and Scarisbrick R ( 1951) The haematin compounds of leaves. New Phytol 50:98--111 Hippler M, Ratajezak R and Haehnel W (1989) Identification of the plastocyanin binding subunit of Photosystem I. FEBS Lett 250: 280-284 Huzisige H and Ke B (1993) Dynamics of the history of photosynthesis research. Photosynth Res 38:185-209 Izawa S, Kraayenhof R, Ruuge EK and Devanlt D (1973) The site of KCN inhibition in the photosynthetic electron transport pathway. Biochim Biophys Acta 314:328-339 Katoh S (1959a) Studies on the algal cytochrome of c-type. J Biochem 46:629-632 Katoh S (1959b) Studies on algal cytochrome. I. Enzymic activities pertaining to Porphyra tenera cytochrome 553 in ceil-free extracts. Plant Cell Physiol 1: 29-38 Katoh S (1960a) Crystallization of an algal cytochrome, Porphyra tenera cytochrome 553. Nature (London) 186:138-139 Katoh S (1960b) Studies on algal cytochrome.II. Physico-chemical properties of crystalline Porphyra tenera cytochrome 553. Plant Cell Physiol 1:91-98 Katoh S (1960c) A new copper protein from ChloreUa ellipsoidea. Nature (London) 186:533-534 Katoh S (1961a) Inhibitory effect of light on oxygen-uptake by cell-free extracts and particulate fractions of Rhodopseudomonas palustris. J Biochem49:129-132 Katoh S (1961b) Flavoprotein in Chlorella eUipsoidea catalyzing TPNH-llnked reduction of plastocyanin. Plant Cell Physiol 2: 165-173 Katoh S (1972) Inhibitors of electron transport associated with Photosystem II in chloroplasts. Plant Cell Physiol 13:273-286 Katoh S and San Pietro A (1966a) The role of plastocyanin in NADP photoreduction by chloroplasts. In: Peisach J, Aisen P and Bhimberg WE (eds) Biochemistry of Copper, pp 407--422. Academic Press, New York Katoh S and San Pietro A (1966b) Inhibitory effect of salicylaldoxime on chloroplast photooxidation-reduction reactions. Biochem Biophys Res Commun 24:903-908 Katoh S and San Pietro A (1967) Photooxidation and reduction of cytochrome-552 and NADP photoreduction by Euglena chloroplasts. Arch Biochem Biophys 121:211-219 Katoh S and Takamiya A (1961) A new leaf copper protein plastocyanin, a natural Hill oxidant. Nature (London) 189:665--666 Katoh S and Takamiya A (1963a) Photochemical reactions of plastocyanin in chloroplasts. In: Kok B and Jagendoff A (eds) Photosynthetic Mechanisms in Green Plants, pp 262-272. Nati Acad Sci-Natl Res Council Publication # 1145, Washington Katoh S and Takamiya A (1963b) Light-induced reduction and oxidation of plastocyanin by chloroplast preparations. Plant Cell Physiol 4:335-347 Katoh S and Takamiya A (1964) Nature of copper-protein binding in spinach plastocyanin. J Biochem 55:378-387 Katoh S and Takamiya A (1965a) The role of plastocyanin in photoreduction of indigo carmine in chloroplasts. J Biochem 58: 396-406 Katoh S and Takamiya A (1965b) Restoration of NADP photoreducing activity of sonicated chloroplasts by plastocyanin. Biochim Biophys Acta 99:156-160

Katoh S, Suga I, Shiratori I and Takamiya A (1961) Distribution of plastocyanin in plants, with special reference to its localization in chloroplasts. Arch Biochem Biophys 94:136-141 Katoh S, Satoh K, Yamagishl A and Yamaoka T (1975) Fluorescence induction in chloroplasts isolated from the green alga Bryopsis maxima. I. Occurrence of the complete Kautsky effect in Bryopsis chloroplasts. Plant Cell Physiol 16:1093-1099 Kimimura M and Katoh S (1972) Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin; inhibitory effects of HgC12 on electron transport and plastocyanin in chloroplasts. Biochim Biophys Acta 283:279-292 Knaff DB and Amon DI (1969) A concept of three light reactions in photosynthesis by green plants. Proc Natl Acad Sci USA 64: 715-722 Kok B, Ruraniski HJ and Harman EA (1964) Photooxidation of cytochrome c, f, and plastocyanin by detergent treated chloroplasts. Plant Physiol 39:513-520 Krauss N, Hinrichs W, Witt I, Fromme P, Printzkow W, Danter Z, Betzel C, W'flson KS, Witt HT and Saenger W (1993) 3Dimensional structure of system I of photosynthesis at 6 angstrom resolution. Nature (London) 361:326-331 Landeg~rdh H (1954) On the oxidation of cytochrome f by light. Physiol Flantarum 7:375-382 Martinez SE, Huang D, Szezepaniak A, Cramer WA and Smith JL (1994) Crystal structure of chloroplast cytochrome f reveals a novel cytochrome fold and unexpected heme ligation. Structure 2:95-105 Michel JM and Michel-Wolwertz MR (1969) On the fractionation of the photosynthetic apparatus of spinach chloroplasts. In: Metzner H (ed) Progress in Photosynthesis Research, Vol I, pp 115-121. Intl Union Bioi Sci, Tiibingen Nakamura H (1937) ~)ber das Vorkommen der Hydrogenlyase in Rhodobacillus palustris und Uber ihre RoUe in Mechanismus der bacterielle Photosynthese. Acta Phytochim 10:211-218 Neish AC (1939) Studies on chloroplasts. II. Their chemical composition and the distribution of certain metabolites between the chloroplasts and the remainder of the leaf. Biochem J 33:300-308 Nieman RH and Vennesland B (1959) Photoreduction and photooxidation of cytochrome c by spinach chloroplast preparations. Plant Physiol 34:255-268 Peterson EA and Sober HA (1956) Chromatography of proteins. I. Cellulose ion-exchange adsorbents. J Am Chem Soc 78:751-755 Sane PV and Hauska GA (1972) The distribution of photosynthetic reactions in the chloroplast lamellar system. I. Plastocyanin content and reactivity. Z Naturforsch 27:932-937 Shibata Ka, Benson AA and Calvin M (1954) The absorption spectra of suspensions of living microorganisms. Biochim Biophys Acta 15:461--470 Shibata Ke (1931) Carbon and Nitrogen Assimilation in Biology (in Japanese) Iwanami, Tokyo. [English translation by Gest H and Togasaki RK (1975) Japan Science Press, Tokyo] Shibata Ke and Yakushiji A (1933) Der Reactions Mechanismus tier Photosyntheses. Naturwissenschaften 21:266-268 Sykes AG (1991) Plastocyanin and the blue copper proteins. Chem Soc Rev 14:283-315 Takamiya A (1963) Hiroshi Tamiya, life and work. In: Studies on Microalgae and Photosynthetic Bacteria. A special issue of Plant Cell Physiol, pp xv-xix. Univ Tokyo Press, Tokyo Tamiya H (1949) Analysis of photosynthetic mechanism by the method of intermittent illumination. II. Theoretic part. In Studies from the Tokugawa Institute, Vol VI, pp 43-129 Tamiya H (1955) Keita Shibata. Bericht Deutsch Bot Geselschaft LXVIIa: 13-16

189 Tamiya H (1957) Mass culture of algae. Ann Rev Plant Physiol 8: 309-334 Tarniya H (1966) Synchronous cultures of algae. Ann Rev Plant Physiol 17:1-26 Tamiya H and Chiba Y (1949) Analysis of photosynthetic mechanism by the method of intermittent illumination. I. Experimental part. In Studies from the Tokugawa Institute, Vol VI, pp 1--42 Tamiya H and Huzisige H (1942) Comparison between photosynthesis and peroxide reaction in respect to their sensitivity towards some poisons (in Japanese) Kagaku 12:329-333 Tamiya H, Iwamura T, Shibata K, Hase E, Nihei T (1953) Correlation between photosynthetic and light-dependent metabolism in the growth of Chlorella. Biochim Biophys Acta 12:23-40 Tamiya H, Miyachl S and Hirokawa J (1957) Some new preillumination experiments with carbon-14. In: Gaffron H, Brown AH, French CS, Livingson R, Rabinowitch EI, Strehler BL and Tolbert NE (eds) Research in Photosynthesis, pp 205-212. Intersci Publ Inc, New York Thunberg T (1923) Ober einen neuen Weg vonder Kohlens~iure zum Formaldehyd. Ein Beitrag zum Problem der Kohlens[iureAssimilation. Z Physik Chem 106:305-313 Trebst A and Eck H (1963) Ober eine p-Hydroxylierung in isolierten Chloroplasten. Z Naturforsch 18:105-109 Van Niel CB (1930) Photosynthesis of bacteria. In: Contributions to Marine Biology, pp 161-169. Stanford University Press, Stanford, and Humpry Milford, Oxford University Press, London Van Niel CB (1941) The bacterial photosynthesis and their importance for the general problem of photosynthesis. Adv Enzymol I: 263-328

Van Niei CB and Muller FM (1931) On the purple bacteria and their significance to the study of photosynthesis. Recueil des Travaux Botanique Neerlandais (JH de Busry, Amsterdam) 28:245-274 Vernon LP and Hobbs MO (1957) Reduction of low-potential compounds by illuminated leaf homogenate and chloroplasts. Arch Biochem Biophys 72:25-36 Vernon LP and Kamen MD (1954) Hematin compounds in photosynthetic bacteria. J Biol Chem 211:643-662 Whatley FR, Ordin L and Arnon DI (1951) Distribution of micronutrient metals in leaves and chloroplast fragments. Plant Physiol 26:414-417 Wood PM (1977) The role of c-type cytochromes in algal photosynthesis. Extraction from algae of a cytochrome similar to higher plant cytochrome f. Ear J Biochem 72:605-612 Wood PM (1978) Interchangeable copper and iron proteins in algal photosynthesis. Studies on plastocyanin and cytochrome c-552 in Chlamydomonas. Eur J Biochem 87:9-19 Wurmser R (1930) Oxydations et r6ductions. Presses Universitaire de France, Paris Yakushiji E (1935) IJber das Vorkommen des Cytochroms in hoheren Ptlanzen und Algen. Acta Phytochlmica 6:325-329 Yakushiji E, Uchino K, Sugimura Y, Shiratori 1 and Takamiya F (1963) Isolation of water-soluble chlorophyll protein from the leaves of Chenopodium album. Biochim Biophys Acta 75: 293298 Yamaoka T, Satoh K and Katoh S (1978) Photosynthetic activties of a thermophilic blue-green alga. Plant Cell Physiol 19:943-954