Light and dark rate-determining steps in electron transport reactions in spinach chloroplasts

Plant & Cell Physiol. 13: 885-897 (1972) Light and dark rate-determining steps in electron transport reactions in spinach chloroplasts Kazuhiko Satoh 1, Sakae Katoh and Atusi Takamiya 1 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo, Japan (Received May 2, 1972) Effects of various factors, such as uncouplers, inhibitors and inhibitory treatments of chloroplasts, on light-dependent (KL) and -independent (KD) parameters estimated from the light intensity-activity relationship, were studied. In the Hill- reaction with ferricyanide or methyl viologen as electron acceptor, a reagent or treatment affecting electron transport on one side of system II, which included the rate-limiting step for the entire electron transport, affected only. In contrast, a change in the rate of electron transfer on the other side of system II affected only. We inferred that represents the rate of the dark rate-limiting step at infinite light intensity. On the other hand, is concerned to the quantum yield of the primary reaction, as well as to the rate constant of the reaction on the side of system II opposite to that of the rate-limiting step at infinite light intensity. Effects of inhibitors and treatments on the reaction parameters changed markedly depending on the of the reaction medium. However, the contrasting effects of inhibitors affecting the opposite sides of system II were consistently observed in denned levels of. This was also the case in the photoreduction of methyl viologen with the ascorbate-dpip couple as electron donor. Lumry, Spikes and Rieske (1-3) showed that there was a simple, rectangular hyperbolic relationship between the Hill reaction rate (V) and the intensity of actinic light (I): where, KL is the rate constant of the rate-limiting light step, and KD the reaction rate at infinite light intensity. Since then, our knowledge of electron transport in photosynthesis has greatly Abbreviations: DPIP, 2,6-dichlorophenol-indophenol; DCMU, 3-(3,4-dichlorophenyl)-l,ldimethylurea; Tricine, N-tris (hydroxymethyl)methylglycine; MES, 2-(N-morpholino)ethancsulfonic acid; Cl-CCP, carbonyl cyanide m-chlorophenylhydrazone; HOQNO, 2-heptyl- 4-hydroxyquinoline-N-oxide; TES, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid. 1 Present address: Department of Biology, Faculty of Science, Toho University, Narashino, Chiba, Japan. 885

886 K. Satoh, S. Katoh and A. Takamiya advanced. There is a body of evidence supporting the hypothesis that two photochemical reactions sensitized by two pigment systems of different pigment composition, pigment system I and II according to Duysens (4, 5), are operating in series in the electron transport system of photosynthesis. At high intensities of light, the rate-limiting step in electron transport in isolated chloroplasts lies at a site between the two photosystems, where it is coupled with the phosphorylative reaction (6); thus, the Hill reaction rate can be enhanced by adding phosphorylating reagents or uncouplers. The rate of the Hill reaction can also be controlled by a wide variety of inhibitors of photosynthetic electron transport. Many inhibitors, as exemplified by o-phenanthroline and DCMU, have been shown to inhibit electron transport on the reducing side of system II (cf. Ref. 4). New ways have recently been discovered to control electron transport on the oxidizing side of system II, i.e., heat-treatment (7) and Tris-washing (8) of chloroplasts. A wide variety of inhibitors (9, 10) also preferentially suppress electron transport from water to system II. The inhibited electron flow through system II in heat- or Tris-treated chloroplasts was shown to recover on adding artificial electron donors, e.g. ascorbate, Mn ++ or phenylenediamine-ascorbate couple (7, 11). We studied the effects of inhibitors and inhibitory treatments of chloroplasts on the Hill reaction and related light-induced oxidation-reduction reactions under various intensities of illumination. Changes in the reaction parameters, KD and KL in equation 1, induced by the inhibitors and inhibitory treatments depended on which side of electron transport (with respect to system II) was rate-limiting in the chloroplast preparation used and was affected by the inhibitors or the treatments. KD is shown to be the rate-limiting step in electron transport under infinite light intensities and KL is related to the efficiency of the primary photoact, as well as to the rate of electron transport on the non-rate-limiting side of system II. Materials and methods The source of chloroplasts was fresh spinach leaves obtained from a local market. Chloroplasts were prepared as described previously {12), except that the washing medium contained 0.1M sucrose and 5 ITIM TES-KOH ( 7.4). EDTA-treatment of chloroplasts was carried out as described by Izawa et al. (13). Hill activity with ferricyanide as electron acceptor was determined spectrophotometrically, by measuring the absorbance decrease at 420 nm with a Hitachi spectrophotometer EPU 2A, modified as described elsewhere (14). The reaction mixture contained, in a final volume of 2 ml; 25 ITIM tricine-koh (or MES-KOH) buffer of the indicated, 0.1 M sucrose, 10 ITIM KC1 and the indicated amounts of Hill oxidant and chloroplasts. When evolution or uptake of oxygen was determined, a Clark type oxygen electrode was used, as described elsewhere (15). The reaction mixture was the same as above, but the total volume used in the measurement was 4 ml. Red light (>660 nm) from a 500 W Ushio quarz-iodine lamp passed through a 7 cm layer of water and a red cut-off filter, Mitsubishi VR66, was used as the actinic light in spectrophotometric measurements. Light intensity designated as "1" in the figures was approximately 4-10 5 ergs/cm 2 -sec.

Rate-limiting steps of the Hill reaction 887 Actinic light in the (^-measurement was red light (>600 nm) from a 300 W tungsten lamp. Light intensity designated as "1" in the figures was 2-10 5 ergs/cm 2 -sec. All experiments were performed at room temperature. Chlorophyll was determined according to the method of Arnon (16). Results Hill reaction with ferricyanide as oxidant Fig. 1 shows the relationship between the intensity of actinic light (I) and the rate of ferricyanide Hill reaction (V) in chloroplasts at 7.0. The doublereciprocal plots from Eq. 1 gave a set of straight lines for the various experimental conditions. In the chloroplasts employed here, the rate of overall electron flow at a strong light intensity was controlled by the phosphorylating reaction associated with the electron transport. In fact, the rate of ferricyanide Hill reaction was greatly enhanced by the addition of an uncoupler, methylamine, to the reaction mixture; thus, reflecting tight coupling of electron transport with the phosphorylation process in chloroplasts. Fig. 1A shows that methylamine caused a lowering of the intercept of the ordina.te with the straight line, corresponding to an increase 30 ' Untreated chloroplasls heal 40"C 2min 2 4 6 I/Lighl intensity (A) 10 EOTA- and heat-treated control 2 4 6 I/Light intensity Fig. 1. Effects of DCMU, methylamine and heat-treatment of chloroplasts on the ferricyanide Hill reaction in untreated spinach chloroplasts (A) and effects of DCMU or Cl-CCP in EDTA- and heat-treated chloroplasts (B) at various light intensities. The reaction mixture contained; 25 mm tricine- KOH ( 7.0), 0.1M sucrose, 10 mm KC1, 0.4 mm ferricyanide, chloroplasts equivalent to 15.7,ug chlorophyll/ml (A) and 20.0 fig chlorophyll/ml (B). In (B), chloroplasts were treated with 0.5 mm EDTA twice, then heated at 40 C for 3 min. Where indicated, 20 nm DCMU, 10 mm methylamine or 2 fit* (A) or 1 /JM (B) Cl-CCP was added. Light-induced oxygen evolution was determined with a Clark type oxygen electrode. (B) 10 12

K. Satoh, S. Katoh and A. Takamiya o 0 12 4 1/Light intensity Fig. 2. Effects of varying concentrations of DCMU on the fenicyanide Hill reaction in EDTA-treated chloroplasts. Chloroplasts uncoupled by EDTA-treatment were used (8.1 fig chlorophyll/ml). Concentrations of DCMU are indicated in the figure. Photoreduction of ferricyanide was determined spectrophotometrically by following the absorbance decrease at 420 nm. Other experimental conditions were the same as in Fig. 1, except that the of the reaction mixture was 8.0. in KD. At a low concentration of 20 nm, DCMU, an inhibitor which blocks electron transport on the reducing side of system II (4), lowered the Hill activity by decreasing only KD. On the other hand, a mild heat-treatment of chloroplasts (e.g., 40 C, 2 min), which has been shown by Katoh and San Pietro (7) to induce preferential inhibition of electron transport on the oxidizing side of system II, resulted in an increase in the slope of the straight line, corresponding to a decrease in. Interestingly, when chloroplasts were treated with EDTA to uncouple the phosphorylative process from electron transport (17) and were then partially inactivated by mild heat-treatment (40 C, 3 min), the effect of 20 nm DCMU on the light intensity-activity curve of the ferricyanide Hill reaction at 7.0 was opposite to what had been observed with untreated chloroplasts. The inhibitor now affected only KL with no significant effect on KD (Fig. IB). Fig. 2 shows the effects of DCMU on the light intensity dependence of the ferricyanide Hill reaction in EDTA-treated chloroplasts at 8.0. DCMU at 10-20 nm affected only KL with no change in KD. When the concentration of DCMU was increased, however, inhibition became apparent not only in KL but also in KD. Gingrass and Lemasson (18) previously showed that CMU affected only the light reaction at a low concentration, but both light and dark reactions at higher concentrations. Therefore, in this study concentrations of inhibitors, or the extent of inhibitory treatment of chloroplasts, were kept sufficiently low or mild so as to avoid this kind of complication. Cl-CCP is known to behave, besides as an uncoupler, as a potent inhibitor of electron transport in chloroplasts (19, 20). Kimimura et al. (9) recently showed that the inhibition site of Cl-CCP in electron transport chain was on the oxidizing side of system II. In contrast to DCMU, the addition of Cl-CCP at a final concentration of 1 ftm to uncoupled and heated chloroplasts at 7.0 caused a decrease in only KD (Fig. IB). When untreated chloroplasts were used, light intensity plots in the presence of Cl-CCP gave a straight line which crossed the control curve. It had a lowered intercept with the ordinate and a steeper slope as compared

Rate-limiting steps of the Hill reaction 889 Fig. 3. Effects of Cl-CCP on the ferricyanide Hill reaction in EDTA-treatcd chloroplasts in the presence of DCMU at various light intensities. Chloroplasts were treated with EDTA (17.8 /ig chlorophyll/ml). Concentrations of Cl- CCP and DCMU were 2 ^IM and 20 nm, respectively. Other reaction conditions were the same as in Fig. 1. 0 2 4 6 I/Light intensity with the control (data not shown). The increase in KD caused by Cl-CCP is explained by the uncoupling of electron transport from phosphorylation. The Cl- CCP-induced decrease in KL may be a result of inhibition of electron transport on the oxidizing side of system II, since a decrease in KL also occurred on heattreatment of chloroplasts, which affected the same side of system II. Data supporting this conjecture are shown in Fig. 3. In this experiment, chloroplasts were uncoupled by EDTA-treatment, and 20 nm DCMU was included in the reaction medium to make the electron transport on the reducing side of system II ratelimiting. A comparison of plots in the presence and absence of Cl-CCP indicates that Cl-CCP caused a decrease only in KL without affecting KD. Note that the modes of change, with respect to KD and KL, induced by the inhibition of electron transport with Cl-CCP was opposite to that induced by DCMU. Table 1 Effects of various inhibitors on the reaction parameters, KQ and K^, of the ferricyanide Hill reaction Inhibitors DCMU Ioxynil HOQ.NO Piericidine A Cl-CCP Salicylaldoxime Antimycin A Azide Hydroxylami ne Reaction parameters affected Untreated or EDTAtreated chloroplasts KL Reaction conditions were the same as those in Fig. 1. Uncoupled and heated chloroplasts

890 K. Satoh, S. Katoh and A. Takamiya Similar experiments were carried out with various inhibitors of the Hill reaction. Results are summarized in Table 1. The inhibitors studied can be divided into two groups on a basis of their actions on KL and KD. The first group, including DCMU, ioxynil, HOQNO and piericidine A, decreased only KD, in both untreated and EDTA-treated chloroplasts; but only KL in uncoupled and heated chloroplasts. The second group includes salicylaldoxime, antimycin A, Cl-CCP and sodium azide. Their effects on the reaction parameters are opposite to those of the first group, affecting only KL in the untreated or EDTA-treated chloroplasts and only KD in uncoupled and heated chloroplasts. Hydroxylamine was an exception in that it decreased only KL both in untreated or in uncoupled and heat-treated chloroplasts. We recently investigated the effects of these inhibitors on electron transport, fluorescence of chlorophyll a, photobleaching of carotenoids (10) and photooxidation of cytochrome b SS9 in chloroplasts (12). The results indicate that all inhibitors in the first group blocked electron transport on the reducing side of system II; whereas, inhibitors in the second group had inhibition sites on the oxidizing side of system II. The behavior of hydroxylamine as an inhibitor of the electron transport associated with system II was unique and complicated. It has been suggested that hydroxylamine attacks the primary electron donor of system II (14, 21). The evident correlation of the inhibition sites of these inhibitors with their effects on the reaction parameters of electron transport indicates that the modes of change of and depend on which side of electron transport, relative to system II, was affected by the inhibitor. Fig. 4 depicts the effects of DCMU and Cl-CCP, on KD and in the ferricyanide Hill reaction with EDTA-treated chloroplasts, derived from a set of light intensity plots obtained at various levels. In the absence of inhibitors, the 1000 g 500 Ko / A 7 0 5 0 / d KL A \ D \ \ \ \ i d control (o) Cl-CCP (4] DCMU lonm (D) 7 Fig. 4. Effects of DCMU and Cl-CCP on the and K h of the femcyanide Hill reaction at various levels in EDTA-treated chloroplasts. The reaction mixture contained; 25 mm tricine-koh ( 6.5-8.5) or MES-KOH ( 5.5-6.5), 0.1 M sucrose, 10 HIM KC1, 0.4 mm ferricyanide, chloroplasts equivalent to 7.8 pg chlorophyll/ml; 10 nm DCMU or 2 /*M Cl-CCP where indicated.

Rate-limiting steps of the Hill reaction 891 profile of showed a maximum at 7.8 and a bump on the acidic side of the peak around 6.5. The curve of KL also showed double peaks having maxima at 6.5 and 7.7. The shape of the curve, especially the relative heights of the peak and the bump, varied significantly with the chloroplast preparation used. However, the following features concerning the action of DCMU and Cl- CCP on KD and were consistently observed. DCMU caused an appreciable decrease in KD in the neutral region, but became ineffective on increasing or decreasing the of the reaction medium. On the other hand, DCMU induced a marked decrease in KL in the acidic and alkaline regions, but barely affected KL in the neutral region. The -dependency of the action of Cl-CCP on the reaction parameters was opposite to that with DCMU; Cl-CCP decreased only at a neutral, whereas it affected only KD in the acidic and alkaline regions. Hill reaction with methyl viologen as electron acceptor In the ferricyanide Hill reaction, ferricyanide can accept electrons from illuminated chloroplasts somewhere between the two photosystems, as well as at the reducing side of system I. On the other hand, in the Hill reaction with methyl viologen as electron acceptor, cooperation of the two systems is necessary to transport electrons from water to the electron acceptor (22). Therefore, a comparison of the modes of action of various factors affecting different sites of the electron transport chain was made. Fig. 5 indicates that there is, also in the methyl viologen Hill reaction, a linear relationship between the reciprocals for the rate of light induced oxygen uptake and light intensity. In the reaction at 8.0, mild 20 10 (untreated 1 heated PH8.0 no add +Asc. Light intensity 10 6.5 f untreated \ heated 0 I 2 I Light intensity no add +Asc. Fig. 5. Effects of heat-treatment and the addition of ascorbate on the methyl viologen Hill reaction at 8.0 and 6.5 under various light intensities in ED TA-treated chloroplasts. The reaction mixture contained, in a final volume of 4.0 ml: 25 mu tricine-koh ( 8.0) or MES-KOH ( 6.5); 0.1M sucrose; 10 mm KC1 and EDTA-treated chloroplasts equivalent to 15.6 pg chlorophyll/ml. Where indicated, chloroplasts were heated at 35 C for 5 min or 0.1 miu Na ascorbate was added. Light-induced oxygen uptake was followed with a Clark type oxygen electrode,

892 K. Satoh, S. Katoh and A. Takamiya 400 ^ A \ \ > \. / J -. V -..A \ \ i (control (o) <-CI-CCP 2/uM (A) UDCMU 20nM(a) Fig. 6. Effects of DCMU and Cl-CCP on the K u and of the methyl viologen Hill reaction at various levels in EDTA-treated chloroplasts. Concentration of chloroplasts, 9.7 n% chlorophyll/ml. Other conditions were the same as in Fig. 5. Where indicated, 20 nm DCMU or 2 im Cl-CCP was added. heat-treatment of (EDTA-treated) chloroplasts only resulted in a decrease in KD, and the addition of ascorbate, which is known to act as electron donor for system II (7, 23), only increased KD both in heated and non-heated chloroplasts. In contrast, at 6.5 heat-treatment and the addition of ascorbate only caused decreases in KL both in untreated and heated chloroplasts. Fig. 6 shows the effects of DCMU and Cl-CCP on and in the methyl viologen Hill reaction with EDTA-treated chloroplasts at various levels. In the absence of inhibitor, the curves for KD and KL were both double-peaked, having maxima at 6.8 and 7.8 for KD and at about 6.1 and 7.5 for KL. Cl-CCP affected KD at values higher than 7.5 and KL in the neutral and the acidic regions. The effects of DCMU on KL and KD were exactly opposite to those of Cl-CCP with respect to the dependency of the action. DCMU affected in the acidic and neutral regions and KL in the alkaline region. In this reaction, two factors, one affecting electron transport at the oxidizing side and the other at the reducing side of system II showed opposite modes for affecting KD and KL. Although the data are not presented here, both DCMU and Cl-CCP decreased only KL in the DPIP Hill reaction with no effect on in any of the regions tested. This was the only exception from the general rule of the opposite action of these two inhibitors towards KD and KL. Photoreduction of methyl viologen with reduced DPIP as electron donor In the presence of 10,UM of DCMU, photoreduction of methyl viologen with the DPIP-ascorbate couple as electron donor is mediated solely by system I (22). A linear relationship between reciprocals for the photoreduction rate and actinic light intensity also held in this system I-reaction. Since no specific inhibitor of the electron transport associated with system I was known, the flow of electrons on the electron accepting and electron donating sides of system I was varied by changing the concentrations of DPIP and methyl viologen, respectively. 7

Rate-limiting steps of the Hill reaction 893 KO MV=O.lmM (A) MV=0.05mM - 500 10 EDTA-lroaled chits 600 «> 400 * 200 Ko DPIP O.ImM. / / 7 8 DPIP 0.025mM 9 5 7 8 9 20 10 KL DPIP-O.O25mM DPIP=0.lmM EDTA-treated chits 5 6 7 8 9 5 6 7 8 9 Fig. 7. Effects of varying concentrations of methyl viologen (A) and DPIP (B) on the and X L of the methyl vioiogen photoreduction at various levels with the DPIP-ascorbate couple as electron donor. The reaction mixture contained, in a final volume of 4 ml, 25 mu MES-KOH ( 5.5-6.5) or tricine-koh ( 6.5-8.5), 0.1 M sucrose; 10 miu KC1; 0.5 mm ascorbate; 10 /IM DCMU; and chloroplasts equivalent to 5.6 /ig chlorophyll/ml (for A) and 7.7 fig chlorophyll/ml (for B). DPIP concentration was 0.1 mm in (A), and 0.025 mm or 0.1 mm in (B). Methyl viologen concentration was 0.05 mm or 0.1 mm in (A), and 0.1 mm in (B). Fig. 7 shows the values of KD and KL determined for various ranges in the presence of varied concentrations of methyl viologen and DPIP. A comparison of these figures shows that in the alkaline region, where a change in methyl viologen concentration caused a change only in KD, a change in DPIP concentration affected only KL. In the acidic region, changes in the concentrations of methyl viologen and DPIP caused changes only in KL and KD, respectively. Thus, this relatively simple reaction, in which only system I was involved, showed that changes in the electron transport rate on opposite sides of system I exert opposite effect^ on Kp and KL. (B)

894 K. Satoh, S. Katoh and A. Takamiya Discussion The results of this study indicate that the relationship between light intensity and the rates of various light-induced oxidation-reduction reactions in chloroplasts can be approximated by a simple equation for a rectangular hyperbola, with two reaction parameters, KD and KL (Eq. 1), as introduced by Lumry et al. (1-3), who worked on a model of the Hill reaction, including only one photochemical reaction. The present study further confirms that the rectangular hyperbolic relationship equally applies to light-induced electron transfer reactions of chloroplasts, irrespective of whether either one or both of the photochemical systems are involved in the reaction under investigation. According to the interpretation of Lumry et al. (1-3), KL is related to the efficiency of primary photochemical events, and KD to the limiting dark step in the electron transport chain. KL may be related to various factors, i.e. the fraction of incident light absorbed by photosynthetic pigments, the ratio of the distribution of excitation energy between two pigment systems, the fraction of excitation energy trapped by the reaction center, or the quantum efficiency of the primary photoact in the reaction center of chloroplasts. However, note that KL may also be affected by a change in the dark process; a change in the rate of electron flow induced by means of a chemical reagent or treatment of chloroplasts preferentially affecting some particular reaction site alters the oxidation-reduction balance in the electron transport system and may cause changes in the redox states of the primary electron donor and acceptor in the reaction center which, in turn, gives rise to a change in. The above results of analyses of the changes in KL and KD induced by various factors affecting the rates of electron transport in chloroplasts with ferricyanide or methyl viologen as electron acceptor indicate that the modes of change vary depending on the state of the electron transport system in the chloroplasts used, the of the reaction medium and the nature and concentrations of the inhibitors or uncouplers added, as well as the modes of action of the treatment in the chloroplasts used. The experimental results may be summarized as follows: In chloroplasts in which the rate of electron transport is limited at a site on the reducing side of system II coupled (untreated) chloroplasts (in the absence of phosphorylating reagents or uncouplers) or chloroplasts partially inhibited by DCMU the addition of an inhibitor or uncoupler which affects the rate of electron transport on the reducing side of system II induces a corresponding change in KD without affecting KL. On the contrary, the addition of an inhibitor, electron donor or inhibitory treatment of chloroplasts, which affects electron flow at a site on the oxidizing side of system II, causes a change only in KL. The situation is the reverse with chloroplasts uncoupled by EDTA-treatments then heated, so that the rate-limiting step is now on the oxidizing side of system II. Changes affecting electron transfer at sites on the reducing and oxidizing sides of system II induce a change only in KL and KD, respectively. More generally stated, an effect on electron flow on the rate-limiting side of electron transport, with respect to system II, results in a change only in KD, and that on the opposite side, in a change only in KL. The level of the reaction medium was one of the critical factors affecting the light intensity-activity relationship of electron transport in chloroplasts. Lumry

Rate-limiting steps of the Hill reaction 895 and Rieske (1) previously reported that -dependency of and in the Hill reaction showed a single peak, with maxima at approximately 6.3 for KL and 7.3 for KD. They also noted that the position of KD varied with changes in reaction conditions. Detailed investigation in the present study, however, revealed that the -dependency of KL and KD in uncoupled chloroplasts was complicated by the presence of more than one maxima. The positions and relative heights of these maxima varied with the preparation of chloroplasts and the nature of electron acceptor used. The results of repeated experiments with chloroplasts isolated from spinach leaves at various seasons of the year, however, can be summarized as follows, with respect to the dependency of these reaction parameters of Ahe Hill reaction. There is a peak around 8.0 in both the profiles of KD and KL. The peak of KD was markedly affected by Cl-CCP but not by DCMU, whereas that of KL was much more sensitive to DCMU than to Cl-CCP. According to our interpretation, therefore, the ratelimiting step of the overall reaction of the Hill reaction lies on the water side of system II in uncoupled chloroplasts at an alkaline. In the neutral and acidic regions, the profiles of KL and KD for the methyl viologen Hill reaction showed another peak or bump (KD, ca. 7.0; KL, ca. 6.0). DCMU was effective in decreasing KD but did not affect KL in this region. Cl-CCP acted oppositely. We inferred, therefore, that the rate-limiting step is on the reducing side of system II in the acidic and neutral regions. The situation was more complicated with the ferricyanide Hill reaction than with the methyl viologen Hill reaction at values below 7.0. The profile of KD in the ferricyanide system had an additional peak at 6.5 which was suppressed by Cl-CCP but not by DCMU, indicating that the water side of system II contains the rate-determining step at this acidic. Correspondingly, the decrease in was more marked with DCMU than with Cl-CCP in this range. This difference in the rate-limiting step must be ascribed to a difference in sites of electron transport where the Hill oxidant accepts electrons, since ferricyanide can receive electrons from somewhere between the two photosystems as well as at the reducing side of system I. But the latter is the only place where reduction of methyl viologen takes place. Our discussion is limited to results of experiments in which the electron transport system was modified somewhere around system II. In fact, the action ranges of inhibitors or treatments of chloroplasts examined so far, are limited to this region of electron transport in chloroplasts and no appropriate inhibitor is known to specifically affect the non-cyclic electron transport associated with system I. We, therefore, intended to control the rate of electron flow through system I by changing the concentrations of the electron donors and acceptors involved. In this case, a change in the rate of photoreduction of methyl viologen with DPIP-ascorbate couple as electron donor on the electron donating and accepting sides of system I, induced opposite effects on and - Thus, the same approach used with system II may be adopted to elucidate the mechanisms of electron transport associated with system I. In summary, the kinetic approach employed in this study furnishes a useful method for analyzing the sequence of events in electron transport reactions in

896 K. Satoh, S. Katoh and A. Takamiya chloroplasts; especially for discovering which step of the reaction is rate-determining in a given chloroplast preparation under the experimental conditions being investigated. The only exception deviating from the above general rule was the Hill reaction with DPIP as electron acceptor. DCMU and Cl-CCP decreased only KL in each region tested in this reaction. A detailed kinetic study on the DPIP Hill reaction is in progress. This work was supported by a grant from the Ministry of Education. We also wish to acknowledge with thanks the financial aid kindly supplied by the Yamamotonori Company. References ( 1) Lumry, R. and J. D. Spikes: Chemical-kinetic studies of the Hill reaction. In Research in Photosynthesis. Edited by H. Gaffron, A. H. Brown, C. S. French, R. Livingston, E. I. Rabinovitch, B. L. Strehler and N. E. Tolbert. p. 373-391. Interscience Publishers, New York, 1957. ( 2) Rieske, J. S., R. Lumry and J. D. Spikes: The mechanism of the photochemical activity of isolated chloroplasts. III. Dependence of velocity on light intensity. Plant Physiol. 34: 293-300 (1959). ( 3) Lumry, R. and J. D. Spikes: The mechanism of the photochemical activity of isolated chloroplasts. V. Interpretation of the rate parameters, ibid. 34: 293-300 (1959). (4) Duysens, L. N. M. and H. E. Sweers: Mechanism of two photochemical reactions in algae as studied by means of fluorescence. In Studies on Microalgae and Photosynthetic Bacteria. Edited by Japanese Society of Plant Physiologists, p. 353-372. The University of Tokyo Press, 1962. (5) Amesz, J. and L. N. M. Duysens: Action spectrum, kinetics and quantum requirement of phosphopyridine nucleotide reduction and cytochrome oxidation in the blue-green algae Anacystis nidulans. Biochim. Biophys. Ada 64: 261-278 (1962). (6) Arnon, D. I., F. R. Whatley and M. B. Allen: Triphosphopyridine. nucleotide as a catalyst of photosynthetic phosphorylation. Nature 180: 182-185 (1957). ( 7) Katoh, S. and A. San Pietro: Ascorbate-supported NADP photoreduction by heated Euglena chloroplasts. Arch. Biochem. Biophys. 122: 144-152 (1967). (8) Yamashita, T. and VV. L. Butler: Photoreduction and photophosphorylation with Triswashed chloroplasts. Plant Physiol. 43: 1978-1986 (1968). ( 9) Kimimura, M., S. Katoh, I. Ikegami and A. Takamiya: Inhibitory site of carbonyl cyanide m-chlorophenylhydrazone in the electron transport system of rhe chloroplasts. Biochim. Biophys. Ada 234: 92-102 (1971). (10) Katoh, S.: Inhibitors of electron transport associated with photosystem II in chloroplasts. Plant & Cell Physiol. 13: 273-286 (1972). (//) Yamashita, T. and VV. L. Butler: Inhibition of the Hill reaction by Tris and restoration by electron donation to photosystem II. Plant Physiol. 44: 435-438 (1969). (12) Satoh, K. and S. Katoh: Studies on cytochromes in photosynthetic electron transport system I. Photoreduction and photooxidation of cytochrome A559 by photosystem II in spinach chloroplasts. Plant & Cell Physiol. 13: 807-820 (1972). (13) Izawa, S., R. L. Heath and G. Hind: The role of chloride ion in photosynthesis. III. The effect of artificial electron donors upon electron transport. Biochim. Biophys. Ada 180: 388-398 (1969). (14) Katoh, S., I. Ikegami and A. Takamiya: Effects of hydroxylamine on electron-transport system in chloroplasts. Arch. Biochem. Biophys. 141: 207-218 (1970). (15) Satoh, K., S. Katoh and A. Takamiya: Effects of chloride ion on Hill reactions in Euglena chloroplasts. Plant & Cell Physiol. 11: 453-466 (1970). (16) Arnon, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol. 24: 1-15 (1949).

Rate-limiting steps of the Hill reaction 897 (17) Jagendorf, A. T. and M. Smith: Uncoupling phosphorylation in spinach chloroplasts by absence of cations, ibid. 37: 135-141 (1962). (IB) Gingrass, G. and C. Lemasson: A study of the mode of action of 3-(4-chlorophenyl)-l,ldimethylurea on photosynthesis. Biockim. Biophys. Acta 109: 67-78 (1965). (19) Plengridhya, P. and R. H. Burris: Inhibitors of photophosphorylation and photoreduction. Plant Physiol. 40: 997-1002 (1965). (20) De Kiewiet, D. Y., D. O. Hall and E. L. Jenner: Effect of carbonylcyanide m-chlorophenylhydrazone on the photochemical reactions of isolated chloroplasts. Biochim. Biophys. Acta 109: 284-292 (1965). (21) Bouges, B.: Action de faibles concentrations d'hydroxylamine sur remission d'oxygene des algues Chlorella et des chloroplastes d'epinards. Biochim. Biophys. Acta 234: 103-112 (1971). (22) Black, C. C., Jr.: Chloroplast reactions with dipyridyl salts. Biochim. Biophys. Acta 120: 332-340 (1966). (23) Ikeda, S.: Studies on biochemistry of L-ascorbic acid; XXX. Relation between the oxidationreduction system of L-ascorbic acid and biological hydrogen transport system; NO. 11, on the several factors concerning the photooxidation of L-ascorbic acid by leaf homogenate. Mem. Res. Inst. Food Sci. Kyoto Univ. 18: 57-68 (1959).