The Discovery and Function of Plastocyanin: a Personal Account*

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 January 1995

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 situation had improved by that time. In 1955, I joined Prof. Hiroshi Tamiya's laboratory at the Institute of When I entered the University of Tokyo in 1951, it Botany to begin my graduate studies in plant physiol- was my intention to focus my future studies within ogy. agricultural sciences. The reason was very simple. I I did not see Prof. Tamiya often in the Institute of was born in 1932 in Kobe, the largest port in western Botany because he remained at the Tokugawa Insti- Japan which has very recently been severely damaged tute most of the time (see below) and I was allowed to by earthquake, but my family moved to the country- do anything I wanted. The staff members of the labo- side to avoid the anticipated air raids near the end of ratory were Hirosi Huzisige (University of Okayama) the World War II. The area in which we lived was and Shigehiro Morita (Tokyo University of Agriculture rich farmland with numerous rice paddies. However, and Technology). Morita taught me how to start exper- we suffered from a considerable shortage of food since iments. Among the graduate students were Seikichi agricultural production had fallen catastrophically dur- Izawa (Wayne State University), Shigetoh Miyachi ing the war. It seemed to me at that time that there was (University of Tokyo) and Mitsuo Nishimura (Uni- nothing more important than improving food produc- versity of Kyushu). I learned a lot by watching how tion. they designed and executed their mostly successful but The more I learned at the university, however, the occasionally fruitless experiments. As suggested by more I was attracted by the basic biological sciences. Morita, I started my research by examining the effects Plants became familiar to me through several trips to of light on the metabolism of ~4C-labeled lactate in the mountain areas for the collection of plant specimens purple bacterium Rhodopseudomonas palustris. in my first and second year at the university. The food Scientific activities at the university had been essentially non-existent for some time after the war * Invited and editedby Govindjee. 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 temperature- controlled 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 oxy- Fig. 1. Prof.Keita Shibata (1877-1944). The photograph was gen and an oxidant produced by light for respirato- taken in 1935, two years after the publication of the paper on the inhibitory effectsof hydroxylamineon photosynthesisby Yakushiji ry substrates. This study resulted in my first paper and himself. Courtesyof the late Prof. KozoHayashi, his son-in-law. (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 ble to outline the entire history of such research in my manuscript, said to me 'kabe dane', which means the present limited perspective (see Huzisige and Ke 'you are at an impasse'. To myself, I said silently, 'Oh, 1993 for a perspective on the history of photosynthe- no, I am not, sir', since I had several plans, but did sis research). I shall briefly describe the history of our not dare to voice my opinion to my eminent professor. laboratory at the University of Tokyo because this his- I continued to work with chromatophores for some tory corresponds essentially with the early history of time, but Tamiya's words haunted me. Eventually, I photosynthesis research in Japan. gave up the project and started an investigation into The first paper on the mechanism of photosynthesis cytochromes in algae, which led me to the discovery by Japanese authors had appeared in 1933. The authors of plastocyanin. 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 The early history of photosynthesis research in Imperial University of Tokyo (University of Tokyo Japan after the war) and his student Eijiro Yakushiji (Toho University). In their paper, they showed for the first Govindjee suggested to me that I should describe how time that hydroxylamine is an inhibitor of photosyn- investigations into photosynthesis were initiated and thesis. Because hydroxylamine was known to inhibit developed in Japan in this article since this topic is catalase, this finding led them to the conclusion that all not familiar to scientists elsewhere. It is not possi- 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 Nitro- gen 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). Unfortunate- ly, 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 photosynthe- Fig. 2. Prof.Hiroshi Tamiya (1903-1984). Drawnby a street artist sis. Yakushiji found cytochromes in higher plants and in Istanbulin 1950. Courtesyof Prof. Eiji Hase. algae and extracted a cytochrome from a red alga (Yakushiji 1935). This was the first electron carder of photosynthesis isolated, although no one envisioned gy, namely, synchronization of growth phases, allowed its function in photosynthesis at that time. Yakushi- the cellular life-cycle of the alga to be analyzed in ji was also one of the discoverers of a water-soluble detail (Tamiya et al. 1953; Tamiya 1963). His second chlorophyll protein in Chenopodium leaves (Yakushiji contribution was development of the methods for mass et al. 1963). Nakamura studied photosynthesis of pur- culture of the unicellular alga. He established a method ple bacteria and observed the production of H2 from of outdoor culture suitable for a commercially viable certain bacteria (Nakamura 1937). enterprise (Tamiya 1957). Hiroshi Tamiya (Fig. 2, also see Takamiya 1963) Tamiya's scientific achievements and warm hospi- succeeded Prof. Shibata at the Institute of Botany tality attracted many young scientists from different in 1943. He performed detailed quantitative analy- disciplines. Eiji Hase, a chemist, joined the group after ses of the mechanism of photosynthesis both using the war and later succeeded Tamiya at the Research inhibitors (Tamiya and Huzisige 1942) and intermittent Institute of Applied Microbiology. During the war, light (Tamiya and Chiba 1949a,b). When 14C became Tamiya was asked to investigate the medical effects of available in Japan, he and Shigetoh Miyachi undertook certain dyes by a research branch of the Japanese army. a series of kinetic investigations of different steps in During this project, a young lieutenant by the name of photosynthesis by measuring fixation of CO2 in preil- Kazuo Shibata made the acquaintance of Tamiya and luminated Chlorella cells (Tamiya et al. 1957). became a member of the Tokugawa Institute after Japan Tamiya also served as the director of the Tokugawa was disarmed. One day, Shibata (not to be confused Institute for Biological Research and supervised two with Keita Shibata) was asked to record absorption series of important investigations into the growth of spectra of suspensions of algal cells. This task led him Chlorella there. First, introduction of new methodolo- to develop the opal glass method, which allows absorp- 182 tion spectra of turbid suspensions to be recorded with Although photooxidation of cytochrome f (or a sim- minimum interference from light scattering (Shibata et ilar pigment) in algal cells had been reported by Lun- al. 1955). Later he organized, together with Yorinao deggtrdh (1954) and Duysens (1955), less was know Inoue and Teruo Ogawa, an active research group in the about biochemical properties of cytochromes in algae. Institute of Physical and Chemical Research (RIKEN), I started to work with a cytochrome from the red alga Wako. Porphyra tenera in 1958. Several people said to me, Tamiya moved to the newly founded Research 'You'd better use a material unique to Japan', and oth- Institute for Applied Microbiology at the University ers said more explicitly, 'Don't compete with scientists of Tokyo in 1955, and his position at the Institute abroad. You'll never win'. These statements might of Botany was taken over by Atusi Takamiya. When sound ridiculous now but we were very heavily hand- the Department of Biophysics and Biochemistry was icapped by our poor research conditions at that time. established in 1959, Takamiya's group joined the new Porphyra tenera seemed to me to be an appropriate Department. experimental material since it is a unique and favorite I would like to emphasize that the photosynthe- food (nori) in Japan that was at that time cultivated on sis community in Japan is no longer as dynastic as the sea coast of Tokyo. I assumed that I should be able used to be. I am pleased to note that a large number to work with this organism without any competition. of scientists with different scientific backgrounds are This assumption turned out to be incorrect, however, currently engaged in research in photosynthesis. How- and soon I was involved in a competition with two ever, researchers who are related directly or indirectly groups in Japan (see below). to the Shibata-Tamiya-Takamiya's school still account After I had isolated and purified a c-type for the largest proportion of the community, l cytochrome from the alga, I learned that the cytochrome had already been reported by Yakushi- ji as early as 1935, although he considered the Studies on algal cytochrome c-553 cytochrome with an asymmetric a-band to be a complex of two cytochromes (Yakushiji 1935). I found Kinetic analysis was a favorite tool for studies of pho- that cytochrome c-553 was located in the thylakoids tosynthesis and other biological reactions in Tamiya's and had a redox potential that was higher than that laboratory. I followed this tradition in my studies with of cytochrome c in the mitochondrion, but similar to the purple bacterium, but I began to feel after a while that of cytochrome f in the chloroplast (Katoh 1959a, that this approach left a large 'black box' untouched 1960b). The reduced cytochrome was oxidized by the unless more were known about the biochemistry of the thylakoid fraction in the light but not in the dark (Katoh reactions. In the 1950's, evidence began to accumulate 1959b). A similar cytochrome was found in a wide suggesting that cytochromes are involved in photosyn- variety of algae (Katoh 1959a). I concluded, therefore, thesis. Cytochromesfand b6 were found in chloroplas- that cytochrome c-553 should play a role in photosyn- ts by Hill and coworkers (Hill and Scarisbrick 1951), thesis but not in respiration. and their findings were followed by the discovery of In the early sixties, purification of proteins was not Vernon and Kamen (1954) that photosynthetic bac- a simple task and crystallization of a protein was a teria contain large amounts of c-type cytochromes. success worthy of prompt publication in a journal like Nature. So I proceeded to crystallize the cytochrome. i The followingpeople from Takamiya'sgroup are working in It happened that Prof. Okunuki's group at University the fieldof photosynthesisand in related subjects at the time of this of Osaka and one more group in Japan had started the writing: TetsuyaKatoh (Universityof Kyoto), Ryuji Kanai (Uni- versity of Saitama), Hidehiro Sakurai (Waseda University), Norio same project. Okunuki's group had already succeed- Murata (National Institute for Basic Biology),Ken-ichiro Takamiya ed in the crystallization of several cytochromes using (Tokyo Institute of Technology),Kazuhiko Satoh (I-Iimeji Institute column chromatography with a cation-exchange resin of Technology),Mitsumasa Okada (TohoUniversity), Isamu Ikega- for purification. Because Porphyra cytochrome c-553 mi (TeikyoUniversity), Shigeru Itoh (National Institute for Basic Biology) and myself. It should also be noted that Kimiyuki Satoh is an anionic protein, DEAE-cellulose, an adsorbent is from Prof. Huzisige's laboratoryat the Universityof Okayama with anion-exchange properties which had been syn- and Prof. Kazuo Okunuki, a student of Prof. Keita Shibata, fos- thesized by Peterson and Sober (1956), seemed to me tered the talents of pupils such as Takekazu Horio (Universityof more promising. Because no commercial product was Osaka), Masateru Shin (Universityof Kobe)and Takashi Yamashita (Universityof Tsukuba). 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 ous higher plants, but neither in non-chlorophyllous the chemicals that I used. The adsorbent turned out to tissues, such as roots of carrot and turnip, nor in photo- be an excellent tool for purification of the cytochrome synthetic bacteria (Katoh and Takamiya 1961; Katoh et and I was able to crystallize the cytochrome in a short al. 1961). In leaves, the protein was located in chloro- period of time (Katoh 1960a). Okunuki's group also plasts. These data convinced me that we were on the succeeded in crystallization of the cytochrome at about right track. In view of its localization in chloroplasts the same time, and the third group did much later. and characteristic blue color of the oxidized form, the protein was named plastocyanin (Katoh and Takamiya 1961). Discovery of plastocyanin To my disappointment, none of the inhibitors of copper enzymes that we examined seemed to affect or In the year of 1959, I was given about one kilogram of to interact with the copper of plastocyanin (Katoh and freeze-dried cells of Chlorella ellipsoidea, the product Takamiya 1964). Only harsh treatments, such as incu- of mass culture of the alga at the Tokugawa Institute. bation at pH 2.0, or dialysis against 10 mM KCN at pH Unexpectedly, I was unable to find cytochrome c-553 8.0 for 24 h, were effective in removing copper from in repeated attempts with small amounts of the sam- the protein. These treatments seemed to me too drastic ple. Therefore, I decided to use the whole batch of for application to chloroplasts or algal cells [treatment the cells that remained for extraction of cytochrome with KCN was, however, employed for inactivation c-553. When the extract was passed over a large col- of plastocyanin in situ by Izawa et al. (1973)]. We umn of DEAE-cellulose, a number of fractions ranging also examined the effects of Hg 2+ because involve- from pale yellow to red in color were separated. No ment of a sulfhydryl group in the binding of copper cytochrome c-553 could be detected. However, one of to hemocyanin was being discussed at that time. Hg 2+ the yellow fractions was an NADPH-specific flavopro- induced only very slow bleaching of the blue color of tein (Katoh 1961b), and a red fraction was later found the oxidized protein unless the protein structure had to contain ferredoxin. been loosened by treatment with a high concentration During rechromatography of a yellow fraction, I of urea (Katoh and Takamiya 1964). We concluded, noticed that a green band appeared and then gradu- therefore, that Hg2+ was not a good inhibitor of plas- ally faded into the yellow background. I was curious tocyanin. As will be described below, however, this and repeated the column chromatography of the frac- premature conclusion resulted in a long delay in the tion. The green color reappeared temporarily in some elucidation of the function of the protein. cases but not in others. Eventually, I found that ferri- I had little opportunity to discuss the function of cyanide was effective in keeping the substance in ques- plastocyanin in photosynthesis with scientists abroad tion green and this discovery greatly facilitated purifi- because we were very much isolated from the photo- cation of the substance. The purified substance was synthetic community in the Western countries. Shortly blue in color, resembling laccase, a copper-enzyme, after my discovery of plastocyanin, Martin Kamen vis- which happened to be under investigation in a neigh- ited us and, after a very one-sided discussion, he sug- boring laboratory. The blue substance was indeed a gested that I might write to Robin Hill for his opinion copper-containing protein (Katoh 1960c). As expected about the copper protein, and indeed I did. His answer from its behavior during column chromatography, the was not, however, very encouraging. He wrote that, protein had no affinity for oxygen, but was oxidized after reading my article in Nature, he had also found by ferricyanide and reduced by various reductants. plastocyanin in the plants he was studying. His letter Looking into the literature, I found two lines of evi- ended as follows, 'There are several blue Cu-proteins dence to suggest that copper might be involved in pho- that have been described from different animal tissues tosynthesis. (1) A major portion of the copper in green but here there is no evidence that they are related to leaves is located in chloroplasts (Neish 1939; Whatley the cytochrome system except that cytochrome oxidase et al. 1951), and (2) several copper-specific inhibitors preparations have been found to contain Cu'. Several suppress photosynthesis more strongly than respira- people also suggested to me that plastocyanin might tion in Chlorella cells (Greene et al. 1939). These be one of the blue copper proteins with unknown func- observations impelled me to search for a function of tions, or with no function at all. the copper protein in photosynthesis. We found that Nonetheless, I persisted in my work with plas- the protein was also present in green leaves of vari- tocyanin. I reasoned that since plastocyanin has a 184 redox potential of 370 mV, only 5 mV above that of response. I doubt, however, that the 591 nm response cytochrome f, the functional site of the copper pro- is related to plastocyanin because the magnitude of tein should be close to cytochrome f or photosystem the 591 nm absorbance increase, which corresponds I (PS I). The following observations supported my to about half that of the 518 nm electrochromic band hypothesis. Plastocyanin was photoreduced by thy- shift (de Kouchkovsky and Fork 1964), is at least one lakoids but, when the photoreducing activity was sup- order of magnitude larger than would be expected from pressed by treatment of the membranes with deter- the oxidation of all the plastocyanin molecules present gents, the reduced protein was rapidly oxidized in in chloroplasts. Generally, it is difficult to measure the light (Katoh and Takamiya 1963b). Plastocyanin accurately absorbance changes of plastocyanin in situ also greatly accelerated photooxidation of cytochrome because of overlapping absorption changes as well as c by detergent-treated thylakoids (Katoh and Takamiya its small absorption coefficient. 1963b). Kok et al. (1964) showed that plastocyanin- I became confident in that plastocyanin is an intrin- enhanced photooxidation of cytochrome c is most effi- sic electron carrier functioning between the two pho- ciently sensitized by long wavelength light. Thus, tosystems when we found that inactivation of photore- plastocyanin is oxidized by PS I. Earlier work had duction of the physiological electron acceptor NADP + revealed a soluble and heat-labile factor in chloro- is well correlated with release of the copper protein plasts that stimulated photoreduction of indigo carmin (Katoh and Takamiya 1965b; Katoh and San Pietro with reduced DCIP (Vernon and Hobbs 1957) or pho- 1966a). Disruption of thylakoid membranes by sonic tooxidation of cytochrome c (Nieman and Vennesland oscillation resulted in a strong inhibition of NADP + 1959). Plastocyanin also accelerated photoreduction of photoreduction, either water or reduced DCIP as elec- indigo carmine (Katoh and Takamiya 1963b, 1965a). tron donor, concomitant with the release of plasto- The soluble factor and plastocyanin had the same pat- cyanin, while PS II-mediated photoreduction of ferri- tern of distribution and molecular properties. Only the cyanide or DCIP was resistant to the treatment. This molecular size of the factor appeared to be significantly shows that sonic treatment blocks electron transport smaller than that of plastocyanin, but our determination at or near PS I. The treated membranes were also of the size of the plastocyanin molecule turned out to active in photoreduction of plastocyanin but, when PS be an overestimate. Thus, the factor and plastocyanin II electron transport was blocked by an inhibitor, the were one and the same protein. reduced plastocyanin was photooxidized by the prepa- Several groups of investigators suggested that plas- ration. Furthermore, the lost activity of NADP + pho- tocyanin is reduced by PS II and hence serves as an toreduction with water as electron donor was effective- electron carrier between PSI and PS II (Katoh and ly restored by the addition of plastocyanin to sonicat- Takamiya 1963a; Trebst and Eck 1963; Kok et al. ed membranes. We concluded, therefore, that plasto- 1964; de Kouchkovsky and Fork 1964). Evidence pre- cyanin is an intersystem electron carrier that functions sented was, however, not necessarily convincing. We near PS I. observed photoreduction of plastocyanin by thylakoids One day, I received a telephone call from a man (Katoh and Takamiya 1961, 1963b) but the protein is who wanted to know whether I could speak English. I most likely reduced by PSI rather than PS II in this was not available but Nishimura responded, 'I suppose reaction. Trebst and Eck (1963) suggested the par- he can.' So I was invited to the Symposium on Photo- ticipation of plastocyanin in photosynthetic electron synthetic Mechanisms of Green Plants, organized by transport because salicylaldoxime, an inhibitor of cop- Bessel Kok and Andr6 Jagendorf at the Airlie House, per enzymes, strongly suppressed NADP + photore- Virginia, in 1963 and presented a paper on plastocyanin duction with water as electron donor. We showed later, (Katoh and Takamiya 1963a). This was my first trip however, that salicylaldoxime inhibits PS II electron abroad and I met many distinguished people whom I transport (Katoh 1972) but not plastocyanin (Katoh had previously known only from the literature. I was and San Pietro 1966b). De Kouchkovsky and Fork greatly impressed by their warm attitude to a young (1964) found a light-induced absorbance increase at scientist from the Far East. 591 nm in algal cells and isolated chloroplasts and I stayed in the C. F. Kettering Laboratory, in Yellow attributed it to the oxidation of plastocyanin. They Springs, Ohio, from October 1964 to December 1966, proposed that plastocyanin functions between the two working with Tony San Pietro. One of our results sur- photosystems based upon the observation that PSI and prised me: there was no plastocyanin in the cells of PS II lights had antagonistic effects on the 591 nm Euglena gracilis and cytochrome c-553 connected PS 185

I and PS II in the place of plastocyanin (Katoh and the data from the algal mutants were quite convincing San Pietro 1967). I was confused because at that time I and, therefore, I was very surprised when the late Pro- believed cytochrome c-553 to be cytochromef It was fessor Daniel Arnon's group reported results that cast a only much later that cytochrome c-553 was shown to strong doubt on the role ofplastocyanin as an electron correspond to plastocyanin, and not to cytochrome f donor to P-700. (Wood 1977, 1978; Bohner and BOger 1978). In con- Michel and Michel-Wolwertz (1969) separated PS trast to higher plants, which contain only plastocyanin, I and PS II particles, which corresponded to the stroma algae contain either plastocyanin or cytochrome c-553, thylakoids and grana stacks, respectively, from thy- or both. Thus, Chlorella ellipsoidea has only plas- lakoid membranes that had been disrupted by passage tocyanin, but Euglena gracilis lacks the copper pro- through a French pressure cell. Arnon et al. (1970) tein. 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 Functional site of plastocyanin only a marginal effect. This result was taken as evidence for their hypothesis of three light reactions in In the decade that followed the discovery of plas- photosynthesis (Knaff and Arnon 1969; Arnon et al. tocyanin, several different functional sites of plasto- 1970). They proposed that P-700 accepts electron from cyanin in relation to cytochromefwere proposed. cytochrome f in cyclic electron transport, while plastocyanin functions in linear electron transport from .,I plastocyanin water to NADP + which involves two PS II centers P-700 (1) ""- cytochrome f (sequence (4)). They argued that NADP + photoreduc- P-700 ~-- cytochrome f -.-- plastocyanin (2) tion with reduced DCIP is mediated by PSI (P-700) and, hence, proceeds in the absence of plastocyanin. P-700 ~ plastocyanin ~ cytochrome f (3) The new model invited heated arguments among inves- PS IIa -*-- plastocyanin ~ PS IIb (4) tigators because it challenged the widely held concept P-700 ~ cytochrome f ~-] of only two light reactions in photosynthesis. The func- [ tional 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 The notion that plastocyanin and cytochrome f photosynthesis. donate electrons to P-700 in parallel (sequence (1)) was The results of experiments by other groups of put forward on the basis of the observations that both investigators added to the confusion. Consistent with are photooxidized at similar rates in detergent-treated the observation of Arnon's group, Fork and Mura- thylakoids (Kok et al. 1964). As mentioned above, ta (1971a,b) reported that the PSI particles contained however, Euglena cytochrome c-553 employed in this only a negligible amount of plastocyanin but could oxi- study was not cytochrome f. In sequence (2), plasto- dize cytochrome f rapidly in the light. They claimed cyanin was placed before cytochromefbecause salicy- that electron transport from cytochromefto P-700 does laldoxime abolished the 591 nm absorption change and not involve plastocyanin and that the stimulation of PS the photoreduction of cytochrome f (Fork and Urbach I electron transport by added plastocyanin is an arti- 1965). However, the significance of this observation is fact introduced by disruption of membrane structures. not clear for the reasons discussed above. However, other groups found a substantial amount of Sequence (3) was supported by, among others, plastocyanin in PSI preparations (Baszynski et al. the observation that a plastocyanin-deficient mutant 1971; Arntzen et al. 1971; Sane and Hauska 1972). of Chlamydomonas reinhardtii was inactive in PSI The apparent lack of a requirement for plastocyanin in activities, such as photooxidation of cytochrome f the photoreduction of NADP + or the photooxidation (membrane-bound cytochrome c-553) or photoreduc- of cytochrome f was, therefore, ascribed to the pres- tion of NADP + with reduced DCIP (Gorman and ence of the bound plastocyanin in the PSI preparation. Levine 1966). Addition of plastocyanin restored the Investigations with antisera raised against the copper PSI activities. By contrast, a mutant strain deficient protein also yielded equivocal results as to the func- in cytochrome f was able to photoreduce NADP + in tional site of plastocyanin. The function of the copper the absence of added plastocyanin. I considered that protein was a main topic of a symposium that was orga- 186 nized by the late Professor Gtinter Jacobi at G~Sttingen delay in the elucidation of the function of plastocyanin. in 1971. No consensus was reached after three days of However, our findings were made just in time to settle heated discussion because of the largely contradictory the controversy as to whether photosynthesis involves data available at that time. two or three light reactions. Our data were consistent The controversy was finally settled by an experi- with the two light reaction model and ruled out Arnon's ment in which we originally intended to identify an three light reaction model, which assumes that P- inhibitor of PS II electron transport or oxygen evo- 700 receives electrons directly from cytochromefand lution. I suggested to Mamiko Kimimura, a gradu- plastocyanin functions in a separate electron transport ate student, that she examine the effects of Hg 2+ to chain (sequence (4)). Some time later, David Knaff, determine whether sulfhydryl groups are involved in who had been actively engaged in investigations relat- oxygen evolution. Her results were very surprising ed to the three light reaction hypothesis, told me that to me. Treatment of thylakoids with Hg2+ for sever- after reading our paper he gave up the hypothesis. Vari- al minutes had no effect on PS II electron transport ous studies that followed our experiment also provided but strongly inhibited a PSI reaction, namely, pho- evidence against sequence (4). The three light reaction toreduction of methyl viologen with reduced DCIP as model was consigned to the long history of photosyn- electron donor (Kimimura and Katoh 1972). Hg2+- thetic research. treatment also resulted in the inhibition of the pho- Finally, I shall briefly describe the recent advances tooxidation of cytochrome f, but not that of P-700. in research into the function of plastocyanin (for Thus, it appeared that Hg2+ specifically blocked elec- reviews, see Sykes 1991 and Gross 1993). Subunit tron transport between cytochrome f and P-700, a site III of PSI reaction center complex, the product of at which plastocyanin is considered to function in the the psaF gene, was implicated as the plastocyanin- two light reaction model of photosynthesis. I was excit- binding protein (Hippler et al. 1989, but see Chitnis et ed but at the same time puzzled by this finding because al. 1991). The interaction of plastocyanin with its reac- early studies had shown that Hg 2+ is a poor inhibitor tion partners, cytochrome f and P-700, is electrostat- of plastocyanin (Katoh and Takamiya 1964). Soon it ic in nature and strongly influenced by ionic strength occurred to me that we had investigated the effects of and pH of media. Because plastocyanin from higher Hg 2+ only on the oxidized plastocyanin since we had plants is a negatively charged molecule, its binding used its blue color as a convenient indicator of the cop- to positive charges on the partner molecules was sug- per bound to the protein. If plastocyanin were present gested (Gross 1993). The crystal structure of poplar in the reduced state in chloroplasts and the reduced plastocyanin, which was determined by Colman et al. protein were more susceptible to Hg 2+ than the oxi- (1978), has greatly contributed to elucidation of the dized protein, the observed effect of Hg 2+ could be mechanism of electron transfer between plastocyanin ascribed to the inactivation of the plastocyanin. Such and its reaction partners. The copper atom, which is was indeed the case. In contrast to the results with the ligated to a thiol group of Cys 84, a thioester group of oxidized protein, which is strongly resistant to Hg 2+, Met 92 and two imidazol groups of His 37 and His 87, only a brief incubation was needed for the total replace- is embedded in the protein molecule. Electron trans- ment of copper in the reduced protein by Hg 2+. The fer to and from the copper center, therefore, proceeds Hg2+-inactivated chloroplasts contained only Hg2+- through amino acid residues. There are two potential substituted plastocyanin. Furthermore, inhibition was sites on the protein molecule, which are considered to greatly weakened if the thylakoids were treated with participate in the interaction with the reaction partners. ferricyanide to oxidize the protein prior to treatment Different approaches, in particular, chemical modifi- with Hg 2+. It was concluded, therefore, that Hg2+ cation and site-directed mutagenesis showed that Tyr blocks electron transport by specifically attacking plas- 83 and the surrounding patches of negative charges are tocyanin. Thus, the functional site of plastocyanin was involved in the binding of plastocyanin to cytochrome finally established to be between cytochrome fand P- f. Another site is His 87, which separates the cop- 700. per atom from the medium. Electrons are suggest- It took twelve years for me to identify plastocyanin ed to be transferred from copper via an outer-sphere as the primary electron donor of PSI since my dis- mechanism through the coordinated imidazole group covery of the copper protein in 1960. Our premature of this residue (Coleman et al. 1978). Recently, Mar- conclusion that plastocyanin is not very sensitive to tinez et al. (1994) have crystallized and obtained the Hg 2+ (Katoh and Takamiya 1964) resulted in a long structure of the hydrophilic portion of cytochrome f 187 from turnip chloroplasts. The three dimentional struc- numerous investigators. I feel that I have been very for- ture of PSI reaction center complex from the ther- tunate to have been able to witness the amazing devel- mophilic cyanobacterium Synechococcus elongatus is opment of our understanding of photosynthesis during under investigation (Krauss et al. 1993). I expect that this exciting period of time. I have really enjoyed these our understanding of the mechanism of electron trans- 40 years. port in the plastocyanin region will be greatly advanced in the near future. Acknowledgement

Epilogue I thank Govindjee for carefully editing this perspective. 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 References University of Tokyo in 1993, I had to review my career as a scientist because a retiring professor is expected Arnon DI, Chain RK, McSwaln BD, Tsujimoto HY and Knaff DB (1970) Evidence from chloroplast fragments for three photosyn- to give an overview of his achievements in his last thetic light reactions. Proc Natl Acad Sci USA 67:1404-1409 lecture of the university. Preparing for the lecture, I Arntzen CJ, Dilley RA, Peters GA and Shaw ER (1971) Photochem- found that I could recall many experiments that I had ical activity and structural studies of photosystems derived from done 30 years ago with greater clarity than the more chloroplast grana and stroma lamellae. Biochim Biophys Acta 256:85-107 recent ones, perhaps because as a beginning scientist Baszynski T, Brand J, Krogmarm DW and Crane FL (197t) Plasto- everything I did, not only my successes but also my cyanin participation in chloroplast Photosystem I. Biochim Bio- failures, was so new and exciting to me. Since the phys Acta 234:537-540 audience of my final lecture consisted mostly of young Bohner H and B0ger P (1978) Reciprocal formation of cytochrome c-553 and plastocyanin in Scenedesmus. FEBS Lett 85:337-339 people, I spent the first half of my lecture talking about Chitnis PR, Purvis D and Nelson N (1991) Molecular cloning and my early research. What I have written here is most- targeted mutagenesis of the gene psaF encoding subunit III of ly based upon this part of my lecture. The occasion Photosystem I from the cyanobacteriumSynechocystis sp. PCC gave me a chance to count all my past collaborators: 6803. J Biol Chem 266:20146-20151 Colman PM, Freeman HC, Guss JM, Murata N, Norris VA, Ramshaw I was surprised to find that I have had more than sev- JAM and Venkatappa MP (1978) X-ray crystal structure analysis enty coauthors on my papers. They are or were mostly of plastocyanin at 2.7 ,~ resolution. Nature (London) 277:319- young and able investigators. I would like to take this 324 de KouchkovskyY and Fork DC (1964) A possible functioning in opportunity to express my hearty appreciation to all of vivo of plastocyanin in photosynthesis as revealed by a light- them for our joyful and inspirational collaboration. I induced absorbance change. Proc Natl Acad Sci USA 52: 232- have also been impressed by the tremendous changes 239 that have occurred in the environment that surrounds Duysens LNM (1955) Role of cytochrome and pyridine nucleotide in algal photosynthesis. Science 121:61-79 science in Japan since I started my first experiment in Fork DC and Murata N (1971a) Oxidation-reduction reactions of an ill-equipped underground laboratory. I have touched P700 and cytochromef in fraction 1 particles prepared from briefly on the difficult situation that we found during spinach chloroplasts by French press treatment. Photochem Pho- the post-war period, thinking that it might be interest- tobiol 13:33-44 Fork DC and Murata N (1971b) Photochemically-activeparticles ing to young scientists, in particular, to those working from chloroplasts fragmented in a French pressure cell. In: Forti with insufficient facilities in developing countries. G, AvronM and Melandri A (eds) Proc 2nd Intl Congr Photosynth Research into photosynthesis has advanced with Res, Vol I, pp 847-857. Dr. Junk Publishers, The Hague ever-increasing speed since I joined the Tamiya's group Fork DC and Urbach W (1965) Evidence for the localization of plastocyanin in the electron-transport chain of photosynthesis. in 1955. Major discoveries have been followed by more Proc Natl Acad Sci USA 53:1307-1315 major discoveries and the mechanism of this highly Gest (1993) History of concepts of the comparative biochemistry sophisticated and efficient system of plants has been of oxygenic and anoxygenicphotosynthesis. PhotosynthRes 35: elucidated to an extent that no one could have imag- 87-96 Gorman DS and LevineRP (1966) Photosyntheticelectron transport ined forty years ago. To me, photosynthesis research chain of Chlamydomonas reinhardi VI. Electron transport in in the past four decades has been a fascinating dra- mutant strains lacking either cytochrome c-553 or plastocyanin. ma full of new ideas and discoveries, interwoven with Plant Physio141:1648-1656 successes and failures, and joy and disappointments of 188

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