FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE DYNAMICS OF PHOTOSYSTEM I IN GREEN-PLANT PHOTOSYNTHESIS-I. EFFECTS OF ACCEPTORS AND DONORS IN SUBCHLOROPLAST PARTICLES*

Abstract —Transient decay kinetics of Signal I in spinach subchloroplast particles have been studied in the presence of artificial donors [5-methyl phenazonium methyl sulfate (PMS), 2, 6-dichlorophenolindophenol (DPIP), N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD) and ascorbate], artificial acceptors [benzyl viologen (BV) and methyl viologen (MV)] and various natural donor and acceptor moieties, using the technique of flash photolysis-electron spin resonance spectroscopy. The results are discussed in the framework of three mechanisms for reduction of P700⁺: (1) direct electron return from the primary acceptor to P700⁺, (2) cyclic electron flow via an electron carrier, and (3) noncvclic electron flow.     

A LASER FLASH SPECTROSCOPY STUDY OF THE KINETICS OF ELECTRON TRANSFER FROM SPINACH PHOTOSYSTEM I TO SPINACH AND ALGAL FERREDOXINS

Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from P700 in Photosystem I (PSI)-enriched particles from spinach to the ferredoxins from spinach and the green alga Monoraphidium braunii. Very similar behavior for the interaction of both ferredoxins with the PSI complex was observed, although the algal ferredoxin appears to be slightly more effective as an electron acceptor. For both proteins, a non-linear protein concentration dependence of the rate constant for reduction was obtained, indicating complex formation preceding electron transfer. Estimates of 3 times 10⁷M^?1 s^?1 and 140–180 s^?l were obtained from these data for the second order rate constants for complex formation, and the limiting first order rate constants for electron transfer, respectively. At neutral pH, a biphasic dependence of the rate constant for ferredoxin reduction on the concentration of NaCl or MgCl₂ was observed. This was interpreted in terms of the electrostatic interactions which occur between ferredoxin and the PSI membrane. In addition, magnesium cations appear to play a specific role in the interaction between PSI and ferredoxin. Thus, the addition of these ions under optimal conditions induces a 6-f-old increase in the electron transfer reaction rate constant, compared with a 2-f-old increase in the presence of an optimal amount of NaCI. This cannot be explained as arising from ionic strength effects. To our knowledge, this is the first time that a direct measurement of the rate constant for the reduction of ferredoxin by the PSI complex has been reported.     

FLUORESCENCE SPECTROSCOPY OF A P700-CHLOROPHYLL-PROTEIN COMPLEX*

Abstract. Chlorophyll-protein complexes enriched in the Photosystem I reaction center chlorophyll (P700) exhibit a fluorescence emission maximum at 696 nm at - 196°C The height of this 696 nm emission relative to the emission at 683 nm from antenna chlorophyll a increases proportionally with the P700 concentration while the total fluorescence yield of the complex decreases. The 696 nm emission could possibly be from an absorbing form of antenna chlorophyll a that may be somewhat enriched along with P700 in Photosystem I fractions. However, evidence resulting from glycerol treatment which appears to decrease the rate of resonance energy transfer between antenna chlorophyll and P700 favors the hypothesis that the emission comes from a photooxidized P700 dimer (Chl⁺-Chl) absorbing near 690 nm. In turn, this fluorescence evidence provides additional support for the model of a P700 dimer involving exciton interaction. Absorption in the wavelength region of 450 nm specifically excites emission at 696 nm from the P700-chlorophyll complex.     

RAPID REDUCTION OF P-700 PHOTOOXIDIZED BY A FLASH AT LOW TEMPERATURE IN SPINACH CHLOROPLASTS

Abstract—Excitation of chloroplasts at low temperature (down to 4.2 K) by short laser flashes causes largely reversible absorption changes which are attributed to the photooxidation of P-700, the primary electron donor of Photosystem I. At temperatures below 100 K the dark re-reduction of P-700⁺ is biphasic. with half-times of about 122μs and 1.7 ms. The relative contribution of the two phases varies with temperature with the fast phase becoming dominant at the lowest temperatures (˜90% at 5 K). The results are interpreted in terms of tunnelling of the electron from one or two primary accepting sites back to P-700, a process which is largely dominant over the process of charge stabilisation.     

CORRELATION BETWEEN DELAYED LIGHT EMISSION AND FLUORESCENCE OF CHLOROPHYLL a IN SYSTEM II PARTICLES DERIVED FROM SPINACH CHLOROPLASTS

Abstract— The induction transient of delayed light of chlorophyll a, excited by repetitive flashes (0.5 ms in duration) and emitted 0.1 - 1.2 ms after the flashes, was measured in system II particles derived from spinach chloroplasts. An uncoupler, gramicidin S, was always added to the particles in order to eliminate the influence of the phosphorylation system on the delayed light and to isolate a direct relationship between the delayed light emission and the primary photochemical reaction, except for the experiments described in the next paragraph. The yield of delayed light emission from the system II particles was found to be about three–times higher than that of chloroplasts on a chlorophyll content basis. System I particles, on the other hand, emitted much weaker delayed light than chloroplasts. Upon intermittent illumination, induction of delayed light in system II particles showed a decrease from the initial rise level to the steady-state level. The initial rise level was the maximum. The fluorescence induction, on the other hand, exhibited an increase from the initial rise level to the maximum steady-state level. The induction of both delayed light emission and fluorescence arrived at their final steady-state levels after the same period of illumination. Induction of delayed light emission was measured under various conditions that changed the oxidation-reduction state of the primary electron acceptor, X, of photoreaction II: by adding an electron acceptor and an inhibitor of electron transport, and by changing the light intensity. The state of A'was monitored by measuring the fluorescence yield. The yield of delayed light emission excited by each flash was found to depend on the amount of oxidized form of X present before the flash. To examine the role of the primary electron donor Y of photoreaction II in delayed light emission, effects of electron donors of photoreaction II such as Mn²⁺, hydroquinone and p-phenylenediamine were investigated. These agents were found to markedly decrease the yield of delayed light emission without altering the pattern of its induction. They had little effect on the induction of fluorescence. These findings are interpreted by a mechanism in which transformation of the reaction center from the form (X^-Y⁺) into (X Y) produces a singlet excitation of chlorophyll a that is the source of millisecond delayed light emission. This reaction is probably non–physiological and must be very slow if compared to the transformation of (X^-Y⁺) into (X^-Y). Since the form (X^-Y⁺) is produced when the excitation is transferred to the reaction center in the form (XY), it is expected in this scheme that the yield of delayed light emission should depend on the amount of the form (X Y) present before the excitation flashes. Electron donors stimulate transformation of the reaction center from (X^-Y⁺) into (X^-Y). Since this reaction competes with the process of delayed light emission, electron donors are expected to suppress delayed light emission.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

PHOTOSYNTHETIC ACTIVITY AND CHLOROPHYLL ABSORPTION SPECTRA OF FRACTIONS FROM THE ALGA,DUNALIELLA*

Abstract— Dunaliella chloroplasts were fractionated according to C. Arntzen et al, Biochim. Biophys. Acta 256 , 85–107, 1972. The initial French-press treatment and differential centrifugation produced Fraction 1 (Fr 1) enriched in photosystem I activity and a heavier Fraction 2 (Fr 2). When Fr 2 was treated with digitonin followed by either gradient or differential centrifugation, two more fractions were recovered: Fr 1 g with a photosystem 1 activity similar to that of Fr 1, and Fr 2 g with very low photosystem II activity. Photosystem II activity was considerably lower in these Dunaliella chloroplasts and fractions than in spinach particles measured under the same conditions, but the relative activities between the fractions were similar to those for spinach. Fr 2 always had greater photosystem II activity than Fr 1, but the digitonin fractions were low and similar in photosystem II activity. Photosystem II activity was measured as the reduction of 2, 6–dichlorophenol indophenol (DCIP) with H₂O, diphenylcarbazide (DPC) or Mn²⁺ as electron donor. The results indicated that exogenous manganous ion competed with H₂O as an electron donor to photosystem II in broken chloroplasts initially, but after 10–15 s of illumination, the Mn³⁺ formed began to reoxidize DCIP and a cyclic reaction ensued. DPC and Mn²⁺ appeared to react at different sites. Computer-assisted curve analysis of the absorption spectrum of each fraction revealed four major component curves representing the absorbing forms of chlorophyll a at 663, 670, 679 and 684 nm seen in numerous other in vivo chlorophyll spectra (C. S. French et al., Plant Physiol. 49 , 421–429, 1972). However, Fr 2g had approx. 20 percent more of Ca663 and Ca670 and 10% more absorption by chl b than Fr 1 which correlated with the difference in photosystem II activity. On the long wavelength side, Fr 2 g had no Ca694 and almost no photosystem I activity. The results are not sufficient to answer the question of whether the photosystem I particle obtained from the original homogenate is significantly similar to or different from the corresponding fraction obtained from Fr 2 with digitonin.     

INHIBITION OF ELECTRON TRANSPORT IN CHLOROPLASTS BY CHAOTROPIC AGENTS AND THE USE OF MANGANESE AS AN ELECTRON DONOR TO PHOTOSYSTEM II*

Abstract— Incubating spinach chloroplasts with various chaotropic agents results in inhibition of photosynthetic electron transport between water and Photosystem II similar to the inhibition caused by washing chloroplasts with a high concentration of Tris buffer. Partial restoration of NADP photoreduction and fluorescence of variable yield is achieved by adding hydroquinone or Mn²⁺, either of which donates electrons to Photosystem II in the inhibited chloroplasts. The inhibitory treatments cause the release of Mn from its bound state in the chloroplast, thus allowing the measurement of the ESR signal of Mn²⁺. The ESR measurement is used to follow the photooxidation of Mn²⁺ as it donates electrons to photosystem II.     

ANALYSIS OF MICROSECOND FLUORESCENCE YIELD AND DELAYED LIGHT EMISSION CHANGES AFTER A SINGLE FLASH IN PEA CHLOROPLASTS: EFFECTS OF MONO- AND DIVALENT CATIONS

Abstract. New results are presented on the effects of mono- and divalent cations on concurrent changes in the microsecond yields and kinetics of chlorophyll a fluorescence and delayed light emission, and the light saturation curve for the latter at 100 μs, following a 10 ns flash at 337 nm. (1) The fluorescence yield increases exponentially from 3 to 30 μs (lifetime, τ, 6.4 ± 0.6/μs), and decays biphasically between 50 and 800μs. (2) The delayed light emission decays biphasically with two exponential phases: fast phase, T= 7–10μs, and slow phase, T= 33–40μs. (3) The light saturation curve for 100μs delayed light emission is satisfactorily represented by a one-hit Poisson saturation curve. (4) Addition of 5 mM NaCl to salt-depleted chloroplasts decreases (by as much as 40%) the yields of μs fluorescence and delayed light emission, and the subsequent addition of 5mM MgCl₂ increases the yields (≤2 × over samples with only NaCl). (5) The fluorescence yield rise and delayed light emission decay kinetics are independent of low concentrations of cations. The lifetime of the fast phase of fluorescence decay changes from ?90μs to ?160μs, when Na⁺ or Na⁺+ Mg²⁺ are added. Based on a detailed analysis presented in this paper, the following conclusions regarding the effects of low concentrations (few mM) of mono-and divalent cations in sucrose-washed chloroplasts at room temperature are made: (a) Na⁺ decreases (?6%) and Mg²⁺ increases (? 20% compared with the Na⁺ sample) the sensitization of photosystem II photochemistry: this effect is small, but significant. (b) Na⁺ increases and Mg²⁺ decreases the efficiency for radiationless transitions in singlet excited Chl a in the antenna and closed reaction center of PS II; this includes non-radiative energy transfer to PS I, intramolecular intersystem crossing and internal conversion. The ratio of the sum of the rate constants for radiationless transitions to that for fluorescence increases by ? 2-fold upon the addition of Na⁺, and is completely reversed by the addition of Mg²⁺. (c) The rate constant for the re-oxidation of Q^- decreases (about 50%) in the presence of Na⁺ or Na⁺+ Mg²⁺. These conclusions imply that cations produce multiple changes in the primary photoprocesses of PS II at physiological temperatures. It is proposed that these changes are mutually independent and can co-exist.     

STRUCTURE OF PHOTOSYSTEM I AND PHOTOSYSTEM II OF PLANT CHLOROPLASTS*,†,§

Abstract— The action of Triton X-100 upon photosynthetic membranes which are devoid of carotenoids produces a small Photosystem I particle (HP700 particle) which is active in N ADP photoreduction and has a [Chl]/[P700] ratio of 30. The properties of the HP700 particle indicate that it is a reaction center complex which is served by an accessory complex containing the additional light-harvesting chlorophyll of Photosystem I as well as the cytochromes and plastoquinone. When Photosystem II particles obtained by the action of Triton X-100 are further washed with a solution 0.5 M in sucrose and 0.05 M in Tris buffer (pH 8.0), chlorophyll-containing material is released. After centrifugation, the supernatant contains about 1 per cent of the chlorophyll and contains three types of particles which can be separated by sucrose density gradient centrifugation. One of these particles, designated TSF-2b, has the same pigment composition as the original Photosystem II fragment, contains cytochrome 559, and shows Photosystem II activity (DCMU-sensitive diphenylcarbazide-supported photoreduction of 2,6-dichlorophenolindophenol). The other two particles (TSF-2a and TSF-2a′) have a [Chl a]/[Chl b] ratio of 8, have a low concentration of xanthophylls, and show a [Chl]/[Cyt 5591 ratio of about 20. Only the TSF-2a particle is active in the Photosystem II reaction described above. On the basis of these data, it is proposed that the Photosystem II unit consists of a reaction center complex which contains Chl a, Cyt 559, and an acceptor for the photochemical reaction. The reaction center complex would be served by an accessory complex which contains the light-harvesting pigments, Chl a. Chi b, and xanthophyils.     

PHOTOCHEMISTRY OF THE REACTION CENTERS OF SYSTEM II UNDER REPETITIVE FLASH GROUP EXCITATION IN ISOLATED CHLOROPLASTS

Abstract— Absorption changes induced in isolated chloroplasts by excitation with repetitive flash groups have been measured at 690 nm, indicating the photochemical turnover of chlorophyll-a_II (Chl-αn), and at 480 nm and 513 nm respectively, reflecting via electrochromic effect the formation of a transmembrane electric field. The data are compared with measurements of oxygen evolution. In chloroplasts with practically fully intact oxygen evolving capacity it was found: 1. The initial amplitude of the 690 nm absorption change induced by the second flash as a function of the time t_v between the first and second flash of a group increases with a half life of about 35 µs. On the other hand, the average oxygen yield due to the second flash as a function of the time t_v rises with a half life of about 600 µs (and a kinetics in the ms-range of minor extent), confirming the data of Vater et al. (1968). 2. Under far red background illumination, where contributions due to PS I in the µs-range can be excluded, the difference spectrum in the red of the absorption changes induced by the first flash corresponds with that of the absorption changes induced by the second flash fired 200 µ after the first flash. 3. The pattern of the absorption changes at 690 nm induced by repetitive double flash groups at t_v= 200 µs does not markedly change in normal chloroplasts by the presence of DBMIB?. Similar 690 nm absorption changes occur in trypsin treated chloroplasts, independent of the presence of DCMU. 4. The fast regeneration in the µs-range of Chl-a_n is also observed in the third flash of a triple flash group at a time t_v= 200 µs between the flashes of the group. 5. The initial amplitudes of the absorption changes with a decay kinetics slower than 100 µs induced by the second flash at 480 nm and 513 nm, respectively, as a function of the time t, between the first and second flash of a group, are characterized by a recovery half-time of about 600 µs, confirming earlier measurements at 520 nm (Witt and Zickler, 1974). On the basis of these results it is inferred that there does exist a photoreaction of Chl-α_n., with an electron acceptor, referred to as X_a, other than the ‘primary’ plastoquinone acceptor X320, if X320 persists in its reduced state. Under conditions of X320 being in the reduced state, this photochemical reaction was shown to be highly dissipative with respect to charging up the watersplitting enzyme system Y. Furthermore, this Chl-a_n-photoreaction with X_a does not lead to a vectorial transmembrane charge separation, which is stable for more than a few microseconds. Different models for the functional and structural organization of PS II are discussed.     

OXIDATION-REDUCTION REACTIONS OF P700 AND CYTOCHROME f IN FRACTION 1 PARTICLES PREPARED FROM SPINACH CHLOROPLASTS BY FRENCH PRESS TREATMENT

Abstract— –Fraction-1 particles were prepared by passing spinach chloroplasts three times through the French pressure cell and centrifuging in a sucrose gradient. With the electron donor DAD (diaminodurol or 2,3,5,6-tetramethyl-p-phenylenediamine) and ascorbate, a light-induced difference spectrum revealed the oxidation of both cytochrome f and P700 upon illumination of these particles. The oxidation of cytochrome f was completed in less than 0.5 msec. P700 and cytochrome f thus seem to be tightly bound to each other in these particles. Addition of Triton X-100 abolished the fast oxidation of cytochrome f but not that of P700. Artificial electron donors such as DAD, DCIP (2,6-dichlorophenol indophenol), and PMS (N-methylphenazonium methosulfate) were good electron donors for photoreaction 1 in these particles, while neither plastocyanin, Porphyra cytochrome 553, nor Euglena cytochrome-552 reduced P700 efficiently. However, after treatment of fraction 1 particles with Triton X-100 reduced DAD, DCIP and PMS were no longer efficient electron donors, while plastocyanin and the algal cytochromes were highly active in reducing P700. Mammalian cytochrome c was not a good electron donor either before or after Triton treatment. Measurements of the effectiveness of P700 reduction as a function of concentration in Triton-treated particles showed plastocyanin to be about four times more active than Porphyra or Euglena cytochromes which in turn were about fourteen times more active than mammalian cytochrome c. Recent studies by Murata and Brown have shown that plastocyanin is not required for the reduction of NADP in these particles with DCIP and isoascorbate as electron donors. The present investigation and that of Murata and Brown indicate that disintegration of chloroplasts with the French pressure cell and centrifugation in a sucrose gradient is the best method to separate system-1 particles having an electron-transport system in almost the native state as in chloroplasts.     

A STANDARD SYSTEM TO DETERMINE THE QUANTUM YIELD OF SINGLET OXYGEN FORMATION IN AQUEOUS SOLUTION

Abstract —The kinetic behavior of the ESR Signal II in spinach chloroplasts has been studied under steady-state illumination and under flash conditions. In controls Signal II exhibits biphasic decay following cessation of illumination—a moderately fast phase (t_1/2 10-60s) and a slow phase (t_1/2? 2–3 h). Addition of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU), o-phenanthroline, NH₄Cl or gramicidin had no effect on the decay of Signal II; however, agents such as antimycin A, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), NH₂OH or tris washing greatly accelerated the decay of Signal II. Flash photolysis-electron spin resonance experiments on Jensen-Bassham type chloroplasts reveal the presence of a previously unnoticed decay component in the g ? 2 region. This species is formed in less than 1 ms and exhibits a decay half life of ?6-10s. The spectral profile corresponds to the steady-state Signal II spectrum. This new transient is formed in approximately equimolar amounts to Signal I. The results are discussed in terms of two prevalent hypotheses—one which would place the Signal II component on the reducing side of PSII and another which would place the Signal II component on the oxidizing side.     

Destabilization of the Oxygen Evolving Complex of Photosystem II by Al3+

The inhibitory effect of Al³⁺ on photosynthetic electron transport was investigated in isolated thylakoid membranes of spinach. A combination of oxygen evolution, chlorophyll fluorescence induction (FI) and decay and thermoluminescence measurements have been used to characterize photosystem II (PSII) electron transport in the presence of this toxic metal cation. Our results show that below 3 mm , Al³⁺ already caused a destabilization of the Mn₄O₅Ca cluster of the oxygen evolving complex (OEC). At these concentrations, an increase in the relative amplitude of the first phase (OJ) of FI curve and retardation of the fluorescence decay kinetics following excitation with a single turnover flash were also observed. A transmembrane structural modification of PSII polypeptides due to the interaction of Al³⁺ at the OEC is proposed to retard electron transfer between the quinones Q_A and Q_B. Above 3 mm , Al³⁺ strongly retarded fluorescence induction and significantly reduced F_v/F_m together with the maximal amplitude of chlorophyll fluorescence induced by a single turnover flash. This chlorophyll fluorescence quenching was attributed to the formation of P680⁺ due to inhibition of electron transfer between tyrosine 161 of D1 subunit and P680.     

Mechanism of ionization and initial fragmentation in electron impact mass spectra of 3-halogenobenzanthrones

Electron-impact mass spectra of 3-halogenobenzanthrones (halogen X = Cl, Br, I) were measured and ionization efficiency curves and three kinds of linked-scan spectra were obtained for several fragment ions. The fundamental mechanisms of ionization and initial fragmentation were interpreted by the penetration length of an impacting electron or the density distribution on the molecular surface of a rejected electron and its orbital energy. The apparent ionization energy (IE) of a singly charged molecular ion seems to be the lower one of non-bonding electrons on O or X, and that of a doubly charged molecular ion the sum of three terms, the IE of non-bonding electron on O, that on X and the electrostatic repulsion between two positive charges. Two competing pathways of decomposition from the molecular ion M^+· to an ion [M - CO,- X]⁺ were observed: one is the initial detachment of CO in chloro and bromo compounds and the other is the initial elimination of the iodine atom in the iodo compound. The sequence of these reactions was confirmed by metastable ion analysis with linked-scan spectra and the relative magnitudes of the appearance energies. They can be explained by the driving force of a localized positive charge or unpaired electron on a heteroatom.     

UN NOUVEAU MODELE DES CENTRES PHOTOCHIMIQUES DU SYSTEME II*

Abstract— The amount of oxygen evolved by Chlorella cells or by isolated chloroplasts has been measured after illumination by short saturating flashes. In all conditions, the amount of oxygen evolved by one flash is proportional to the fraction of the photochemical centers susceptible to produce oxygen. If dark adapted algae or chloroplasts are illuminated by a sene of flashes, no oxygen is produced by the first flash. This phenomenon is related to the activation process. If y_n is the amount of oxygen evolved by the nth flash of the sequence, it appears that the sene y_n shows large oscillations with a period 4. These oscillations are completely damped after 4–6 periods and the amount of oxygen evolved by a flash reaches a stationary value. For any value of n. the quantities y_n and y_n+2 are linked by a recurrent relation which is the same for Chlorella cells and spinach chloroplasts. No relation can be found between the terms y_n and y_(n+1). The mathematical properties of the series y_n can be understood if one admits that a two memory process is involved in the photochemical reaction. The results have been interpreted in terms of a new model of the System II photochemical centers. The main characteristics of this model are: (1) Each photochemical center includes two electron donors (Z) and one electron acceptor (Q). (2) The formation of one atom of oxygen requires a two quantum process corresponding to the transfer of two electrons from the same electron donor (first memory). (3) The photochemical center acts as a switch which connects alternately each donor to the acceptor (second memory). The switch process occurs after each photoact with an efficiency of about 85 per cent. Other arguments in favor of this model are obtained from studies of the rate of oxygen production at the onset of a weak illumination.     

INHIBITION AND UNCOUPLING OF ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS BY THE HERBICIDE 4,6-DINITRO-O-CRESOL

Abstract— Dinitrophenols are known to affect photosynthetic electron transfer. It is shown that the widely used herbicide 4,6-dinitro-o-cresol is a potent inhibitor of the Hill reaction in isolated chloroplasts. By studying different parts of Photosystem II dependent electron transport it is indicated that this herbicide inhibits at the same site as 3-(3.4-dichlorophenyl)-l,1-dimethylurea.
However, the Photosystem I dependent Mehler reaction ascorbate/dichlorophenolindophenol→ diquat is stimulated at higher concentrations of the herbicide. This stimulation does not occur when an uncoupler is added to the reaction medium. There is also no stimulation of the ascorbatep-tetra-methyl-p-phenylene diamine → diquat Mehler reaction. This suggests that 4,6-dinitro-o-cresol uncouples electron transport in the Photosystem I dependent Mehler reaction, when added at higher     

PICOSECOND FLUORESCENCE STUDIES OF P700-ENRICHED PARTICLES OF SPINACH CHLOROPLASTS

Dynamic properties of the picosecond fluorescence of highly enriched reaction-center particles of photosystem I (8 - 10 chlorophylls/P700) prepared from spinach have been investigated. The number (N) of photons used to excite chlorophyll molecules per reaction center was controlled between 0.06 and 80. The 1/e lifetime was ca. 25 ps for N 1. which is much shorter than previously measured lifetimes of photosystem I particles. The initial fluorescence intensity saturated at higher excitation intensities (N ≲ 1). This was interpreted in terms of interaction and annihilation among excited chlorophyll molecules which occur almost entirely within the duration of a laser flash. The spectrum-resolved fluorescence decay was faster at 690 than at 680 nm. This implies that two kinds of antenna chlorophylls, apart from and in close proximity to P700, have different lifetimes. Upon heat treatment a component with a much longer fluorescence decay time was observed. The growth of this component upon heat treatment at increasing temperatures showed a correlation with a decrease in the amount of P700 that could be photooxidized.     

LIGHT-INDUCED ABSORBANCE CHANGES IN CHLOROPLASTS AT -196°C*

Abstract— Absorbance changes induced by irradiating chloroplasts at — 196°C were measured in the region of 525–575 nm with a single-beam spectrophotometer. Irradiation at low temperature caused a bleaching at 556 nm due to oxidation of cytochrome b₅₅₉ but little or no change of cytochrome f. There occurred in addition a loss of absorbance at 547 nm and an increase at 543 nm. The bleaching at 547 nrn (and possibly the increase at 543 nm) could be induced chemically with dithionite or borohydride but not ascorbate. Subchloroplast particles with only Photosystem I activity showed no light-induced absorbance changes, while particles containing combined Photosystem I and Photosystem II activities showed the same changes as whole chloroplasts. Scenedesmus mutant No. 11 cells showed no absorbance changes while mutant No. 8 and wild-type cells showed the normal changes. It is concluded that the photooxidation of cytochrome b₅₅₉ and the photoreduction causing the bleaching at 547 nm are both mediated by Photosystem II.     

DETECTION OF RADICAL ION INTERMEDIATES IN FLAVIN-PHOTOSENSITIZED PYRIMIDINE DIMER SPLITTING

Photosensitized splitting of cis-syn- and trans-syn-l,3-dimethyluracil dimers by 2′,3′,4′,5′-tetraacetylri-boflavin in acetonitrile containing a trace of perchloric acid was studied by laser flash photolysis. Protonation of the flavin prior to excitation resulted in excited singlet and triplet states that abstracted an electron from the dimers and yielded the protonated flavin radical (F1H₂′⁺), which was detected by absorption spectroscopy. Electron abstraction by the excited singlet state predominated over abstraction by the triplet state. Approximately one-third to one-half of the excited states quenched by the trans-syn dimer yielded F1H₂⁺, the balance presumably undergoing back electron transfer within the geminate radical ion pair generated by the initial electron transfer. A covalently linked dimer-flavin exhibited very inefficient flavin radical ion formation, consistent with the known low efficiency of dimer splitting in this system. These results constitute the first identification of a flavin radical ion intermediate in photosensitized pyrimidine dimer splitting.