STOICHIOMETRY and KINETICS OF PROTON RELEASE UPON PHOTOSYNTHETIC WATER OXIDATION

Abstract— We studied the magnitude and the rise kinetics of proton release into the interior of thylakoids by flash spectrophotometty with neutral red as pH indicator. Excitation of dark-adapted thylakoids by a series of between 4 and 11 flashes produced a complex pattern of proton release into the thylakoid lumen. Proton release upon each flash was time resolved.
A slow component of proton release oscillated weakly in magnitude with period of two as function of flash number. It exhibited a half-rise time of approximately 20 ms from the very first flash on, and it was abolished by inhibitors of plastohydroquinone oxidation. This component was attributed to the oxidation of plastohydroquinone by PS I via the Cytb₆/f complex.
Additionally, rapid and multiphasic proton release was observed with half-rise times of less than 2 ms which exhibited a pronounced and damped oscillation with period of four as function of flash number. This rapid proton release was attributed to water oxidation. A detailed kinetic analysis suggested that proton release occurred with the following stoichiometry and with the following half-rise times during the transitions S₁ S_i+1 of water oxidation: 1 H⁺(250 μs, S₀→₁): 0 H⁺(S₁→ S₂):1 H⁺(200 μs, S₂→S₃):2 H⁺(1.2 ms, S₃→ S₄→ S₀) . Proton release and proton rebinding upon oxidation and reduction of the intermediate electron carrier Z, respectively, may have influenced the kinetics of the respective proton yields but not the stoichiometric pattern.     

ENERGY TRANSFER AND CYTOCHROME FUNCTION IN A NEW TYPE OF PHOTOSYNTHETIC BACTERIUM*

Abstract— The excitation spectrum for bacteriochlorophyll b fluorescence at 1027 mμ in Rhodopseudomonas sp. NHTC 133 indicates that the efficiency of energy transfer from caro-tenoid to bacteriochlorophyll b is between 27 and 28 %.
Light-induced absorbancy changes in anaerobic whole cells indicated the oxidation of three c -type cytochromes (C-550. 5, C-553, C-558) and one b -type cytochrome or cytochromoid C (C-560). At low light intensities C-553 is the main cytochrome oxidized, while at high light intensities mainly C-558 is oxidized in addition to C-553. The light responses of the heme proteins appear to be similar to those observed previously in purple and green photo-synthetic bacteria. No light-induced shifts in carotenoid absorption bands were detected.
In bacterial extracts C-553 and C-558 are bound to the chromatophores, while C-550. 5 is soluble.     

Temperature dependence of charge recombination in Heliobacillus mobilis

Transient absorption difference spectroscopy was used to study the temperature dependence of the P798+ decay kinetics in heliobacteria. For membrane samples, two components were obtained from the fitting of kinetic traces in the temperature range of 4-29 degrees C. A 3-9 ms component representing the cytochrome (cyt) c oxidation has an activation energy of 33.0 +/- 2.8 kJ/mol. A 12-22 ms component representing either P798+FX- or P798+FA/B- recombination has an activation energy of 15.3 +/- 2.4 kJ/mol. In isolated reaction centers (RC), only one 14 ms component due to P798+FX- recombination was obtained in this temperature range. The Arrhenius plot shows that the recombination rate of this P798+FX- state is temperature independent in the near room temperature range. For RC in the temperature range of 60-298 K, a 12-15 ms decay was obtained at temperatures greater than 240 K. Biphasic decay traces (12-15 ms and 2-4 ms components) were obtained at temperatures between 170 K and 230 K. Only one 2-4 ms component was found at temperatures lower than 160 K. The gradual switchover from the 12-15 ms to the 2-4 ms component upon cooling may indicate the shift of the P798+FX- recombination state to a state that is prior to P798+FX-, although other interpretations can not be excluded. The absorption difference spectrum (delta A @ 160 K - delta A @ 240 K) in the blue region shows a positive amplitude below 405 nm and a negative amplitude above 405 nm implying that the 2-4 ms decay component may be due to the recombination of P798+A1-, where A1 is a quinone-type acceptor.     

MEMBRANE POTENTIAL AND MICROSECOND TO MILLISECOND DELAYED LIGHT EMISSION AFTER A SINGLE EXCITATION FLASH IN ISOLATED CHLOROPLASTS

Abstract— The effect of light-induced and salt-jump induced membrane potential on microsecond and millisecond delayed light emission from chloroplasts, following a single 10 ns flash, have been studied. Microsecond delayed light emission is shown to be independent of the membrane potential contrary to proposals that the activation energy for delayed light emission can be modulated by transmembrane electric fields. This result is discussed in terms of the possible origin of this short-lived emission. Millisecond delayed light after a single excitation flash is enhanced by membrane potential only if a proton gradient is present. By measuring changes in ms delayed light caused by simultaneous injection of KC1 and Na-benzoate (which creates a proton gradient) in the presence of valinomycin, the light-induced potential generated across the thylakoid membrane by a single excitation flash was calibrated and found to be 128 ± 10 mV in agreement with the recent measurements of Zickler and Witt (1976) based on voltage-dependent ionophores. It is concluded that the secondary charges that give rise to ms delayed light, after a single flash, do not fully span the membrane.     

THERMOLUMINESCENCE STUDIES ON SPINACH LEAVES AND EUGLENA

Abstract— Spinach leaves and Euglena cells when frozen in light to 77 K emit light during slow warming in the dark to give 6 peaks. The peak appearing at 118 K is observed even after DCMU or heat treatment and also in aged chloroplasts that are inactive in electron transport.
The data indicate that peaks appearing at 261 and 321 K are due to back reactions of primary acceptors of PS II and PS I respectively with oxidized chlorophylls. The DCMU sensitivity of Tl peaks at 283 and 298 K suggests that they are associated with the flow of electrons between PS II and PS I. Evidence has been presented to show that the PS 1 chlorophylls are involved in part of the luminescence observed during the temperature rise.
A mechanism involving the return of the thermally detrapped electrons to the ground state of chlorophylls through their excited states has been proposed to explain some of the Tl peaks.     

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.     

FAST AND SLOW PHASES OF LUMINESCENCE IN CHLORELLA

Abstract— The decay of luminescence of Chlorella in repetitive steady-state flashing light displays two categories of phase, broadly characterized as ‘fast’ and ‘slow’. They are strongly contrasted in amplitude and lifetime; the transition from fast to slow occurs a few milliseconds after the flash.

• 1 The slow phases observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) are clearly of the deactivation type. i.e. the luminescence intensity is quantitatively correlated with the rate of relaxation of the System II reaction center to its photoactive state. The significance of the deactivation type is a reverse flow of the light-produced ⊕ and ? charges through the luminescence-producing recombination path. This mechanism is probably not limited to the DCMU-poisoned systems. The light regime (flash duration, flashing period, induction effects) modifies specifically the amplitude of the slow phases; this effect is strikingly not dependent on the presence of DCMU. Although the light-driven pH gradient is the likely explanation, it is argued that its action bears more directly on the exciton and/or emission yield of luminescence rather than on the rate of recombination itself.
• 2 Most of the fast-phase components are ascribed under normal conditions to stabilization steps involving the donor side of the System II reaction centers. However, when the reaction center turnover is much reduced (DCMU) or completely abolished (DCMU + NH₂OH, after preillumination), a fast luminescence phase is still visible. This phase is barely affected by the light regime (notably the flash duration). It must be attributed to an anomalous residual photochemical turnover of the reaction centers. Another type of fast photochemical turnover has been characterized with NH₂OH-pretreated cells. A two-quantum functioning of some sort seems required to account for the anomalous photochemical turnover. The importance of the luminescence loss during the fast phases and its possible connection with the ‘misses’ of the O₂-evolving system are discussed.

     

THE TRIPLET STATE OFPYRIDOXAL–5'-PHOSPHATE IN LASER FLASH PHOTOLYSIS

Abstract— Sensitization and quenching experiments demonstrate that laser flash photolysis ofpyridoxal–5'-phosphate populates a triplet state about 244 kJ/mol. Excitation by the 337.1-nm photons from a nitrogen laser generated transient absorption in the 400-nm region which decayed with a fast first order component on a much slower one. Xanthone photosensitized the same absorption; oxygen, nitric oxide, biacetyl, and 1,3-cyclo-hexadiene quenched it. Spectra and lifetimes suggest that the triplet state leads to radical formation.     

Dihydrophenanthrene-type intermediates during photoreaction of trans-4'-benzyl-5-styrylfuran

[structure: see text] Photoreaction of trans-4'-benzyl-5-styrylfuran (trans-BSF) has been studied by the 355-nm laser flash photolysis (LFP) in CH2Cl2 using a Nd3+:YAG laser (30 ps, 5 mJ pulse(-1) or 5 ns, 30 mJ pulse(-1)). Transient fluorescence and absorption spectra assigned to the singlet excited trans-BSF were observed during the 30-ps LFP, whereas a transient absorption spectrum with two peaks at 400 and 510 nm, assigned to the trans-fused dihydrophenanthrene (DHP)-type intermediate (DP1), was observed during the 5-ns LFP. It is clearly suggested that a two-photon absorption process is involved in the formation of DP1. The first photoreaction is the photoisomerization of trans-BSF, which occurs to give cis-BSF. The second photoreaction process is photocyclization of cis-BSF, which occurs to give DP1 decaying with the half lifetime (tau1/2) of 2.8-4.0 micros to produce another DHP-type intermediate (DP2) with an absorption peak at 400 nm in the absence of O2, through [1,9]-hydrogen shift. DP2 decayed with tau1/2 > 500 micros to give the product through aromatization. In O2-saturated CH2Cl2, DP1 decayed with tau1/2 = 250 ns to give a radical intermediate (X) with two peaks at 410 and 510 nm, through hydrogen abstraction of DP1 by O2. X decayed with tau1/2 = 150 micros to give the product through successive hydrogen abstraction.     

LIGHT-INDUCED ABSORBANCY CHANGES IN EIMHJELLEN''S RHODOPSEUDOMONAS*

Abstract— Light induced absorbancy changes in Rhodopseudomonas sp. NHTC 133 are analogous to those observed in other photosynthetic bacteria, but all of the features in the light-induced difference spectrum (except those reflecting cytochrome oxidation) are shifted to greater wavelengths. This general shift is consistent with the fact that the bacteriochlorophyll of this organism (BChl b ) differs from the usual BChl in that its absorption bands are shifted to greater wavelengths.
The singlet excited state of P985 (the counterpart of the P870 of Rhodopseudomonas spheroides ) is not an energy sink relative to the major component of BChl b in vivo . The absorption maximum of P985 is at 985 mp, whereas that of CBhl b in vivo is at 1012 mμ.     

FLUORESCENCE INDUCTION IN ALGAE AND CHLOROPLASTS

Abstract —The first phases of the fluorescence transient elicited by illumination of dark-adapted algae or isolated chloroplasts (biphasic rise φ_***v - φ_I - _P) have previously been shown to be controlled by two quenchers: Q , the primary acceptor of Photosystem 2 interacting with the secondary acceptor pool A ; and R , a non-photochemical quencher which goes into a non-quenching state as A is reduced.
The dependence of the kinetics of φ decay after illumination upon the redox state of A was studied. It is suggested that some of the centres are in a disconnected state, where electron transfer between Q and A is hindered, the amount of such centres being correlated to the reduction state of A . The implications of this hypothesis on the problem of the variation of the Q-A 'equilibrium constant' under different experimental conditions, and on Murata's 'weak light effect' are discussed.
The effect of 3-(3,4 dichlorophenyl)-1,1 dimethylurea (DCMU) on R is shown to depend upon the redox state of A . A DCMU-induced shift of the midpoint potential of R may account for this dependence.
Evidence is given suggesting that the transient reduction of A which occurs in algae during the φ_I-φ_P rise is controlled by an induction process on the acceptor side of Photosystem 1.     

KINETIC ANALYSIS OF THE FLUORESCENCE INDUCTION IN 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA POISONED CHLOROPLASTS

Abstract— The size of the area over the fluorescence rise curve of chloroplasts is a measure of the total number of quanta utilized in photosystem II during the fluorescence induction, while the growth of the area reflects the progress of photochemical events. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the growth kinetics of the area are affected by the reoxidation of the primary acceptor Q ^- with stored oxidizing charges on the donor side of system II.
At low light intensities, a slow component of this back reaction may limit the steady state fluorescence emission. At higher intensities, however, the fluorescence rise is limited solely by photochemical events, although fast thermochemical reactions like the immediate recombination of photochemically separated charges may affect the efficiency of the photochemistry.
A kinetic analysis of the area growth at moderate light intensities revealed that it occurred in two first order phases which were described by the rate constants k _α and k _β. The biphasic nature suggested a sequential two-electron reduction of the primary acceptor Q , or the presence of two different types of photochemical centers in system II. The rate constants were light intensity dependent. They also were affected by changes in pH, by an addition of NH₂OH, or by a preillumination with short flashes prior to addition of DCMU. It is suggested that the pH of the medium, the presence of NH₂OH, and the flash induced state S_n of the water splitting enzyme, control the values of k _α and k _β by changing the rate constants of electron carrier interactions in the reaction center complex, with a resulting modification of the frequency of back reactions between the primary donor and the primary acceptor.     

Optical probes of intradiskal processes in rod photoreceptors. II: Light-scattering study of ATP-dependent light reactions

Rod outer segment (ROS) disks, either stacked or freely floating, respond to flash illumination to yield a specific, ATP-dependent, light-scattering signal AL. In broken ROS AL signals occur only when AD signals have preceded them. The degree to which the preceding AD signal has been completed determines the amplitude of the following AL signal. However, in freshly detached ROS from dark-adapted frogs Al signals with maximal size can be obtained without pre-incubation with exogenous ATP. The energized state, which is restored in broken ROS with the help of ATP, appears to prevail in the living retina and must therefore be considered to be "physiological". AL signals require structurally intact disks. Neither peripheral ROS proteins nor connecting filaments between adjacent disks are necessary. Their structural origin is the same as that of the preceding AD signal, i.e. osmotic disk swelling. AL signals consist of a single slow kinetic component (half-life 10 s at room temperature) and multiphase fast kinetic component (70 ms). The slow phase corresponds to a light-stimulated resumption of ATPase activity (this has been dealt with in a previous paper) whereas the fast component reflects an immediate response of the energized disk to the metarhodopsin I to metarhodopsin II transition. The latter effect is the subject of this paper. A variety of experiments, using different ATPase inhibitors, ionophores and membrane-permeable salts, have been carried out; they are all consistent with notion that AL originates in the disk interior and probes the existence of a proton electrochemical potential difference delta mu (H+) across the disk membrane. A model is presented which can explain all given properties of AL satisfactorily. According to this model the photolysis of rhodopsin causes a proton release in the disk lumen. This, in turn, results in osmotic swelling of the disks, provided that the internal buffer sites have been (at least partially) titrated with protons prior to the flash. Such conditions, i.e. a low internal pH, are provided by the proton transport across the disk membrane, which presumably takes place during the course of the preceding AD signal.     

Evidence for a lack of reactivity of carotenoid addition radicals towards oxygen: a laser flash photolysis study of the reactions of carotenoids with acylperoxyl radicals in polar and non-polar solvents

In this paper, we report the results of a laser flash photolysis study of the reactions of a range of carotenoids with acylperoxyl radicals in polar and nonpolar solvents. The results show, for the first time, that carotenoid addition radicals do not react with oxygen to form carotenoid peroxyl radicals; an observation which is of significance in relation to antioxidant/pro-oxidant properties of carotenoids. Acylperoxyl radicals, generated by photolysis of ketone precursors in oxygenated solvents, display high reactivity toward carotenoids in both polar and nonpolar solvents, but the nature of the carotenoid radicals formed is dependent on solvent polarity. In hexane, acylperoxyl radicals react with carotenoids with rate constants in the region of 10(9) M(-1) s(-1) and give rise to transient absorption changes in the visible region that are attributed to the formation of addition radicals. All of the carotenoids show bleaching in the region of ground-state absorption and, with the exception of 7,7'-dihydro-beta-carotene (77DH), no distinct absorption features due to addition radicals are observed beyond the ground state absorption region. For 77DH, the addition radical displays an absorption band that is spectrally resolved from the parent carotenoid absorption. The rate of decay of the 77DH addition radical is unaffected by oxygen in the concentration range 10(-4)-10(-2) M, suggesting that these resonance-stabilized carbon-centered radicals are not scavenged by oxygen. At low incident laser intensities, the 77DH addition radical decay kinetics are 1st order with k(1) approximately 4 x 10(3) s(-1) at room temperature. The 1st order decay is attributed to an intramolecular cyclization process, which is supported by the substantial negative entropies of activation obtained from measurements of the decay rate constants for different 77DH addition radicals as a function of temperature. No transient absorption features are observed in the red or near-infrared regions in hexane for any of the carotenoids studied. In polar solvents such as methanol, acylperoxyl radicals also react with carotenoids with rate constants in the region of 10(9) M(-1) s(-1), but give rise to transient absorption changes in both the visible and the red/near-infrared regions, where it is evident that there are two distinct species. For 77DH, the addition radical absorption around 450 nm is still evident, although its kinetic behavior differs from its behavior in hexane. For 77DH and zeta-carotene (zeta-CAR) the spectral and kinetic resolution of the various absorption bands simplifies kinetic analysis. The kinetic evidence suggests that addition radical formation precedes formation of the two near-infrared absorbing species, and that the kinetics of the addition radical decay match the kinetics of formation of the first of these species (NIR1, absorbing at shorter wavelengths). The decay of NIR1 leads to NIR2, which is attributed to the carotenoid radical cation. The solvent dielectric constant dependence of the relative amounts of NIR1 and NIR2 formed leads us to speculate that NIR1 is an ion-pair. However, an alternative assignment for NIR1 is an isomer of the radical cation. The results, in terms of the pattern of reactivity the carotenoids display and of the properties of the carotenoid radicals formed, are discussed in relation to the antioxidant/pro-oxidant properties of carotenoids.     

Carotenoid response to retinal excitation and photoisomerization dynamics in xanthorhodopsin

We present a comparative study of xanthorhodopsin, a proton pump with the carotenoid salinixanthin serving as an antenna, and the closely related bacteriorhodopsin. Upon excitation of retinal, xanthorhodopsin exhibits a wavy transient absorption pattern in the region between 470 and 540 nm. We interpret this signal as due to electrochromic effect of the transient electric field of excited retinal on salinixanthin. The spectral shift decreases during the retinal dynamics through the ultrafast part of the photocycle. Differences in dynamics of bacteriorhodopsin and xanthorhodopsin are discussed.     

INTERACTION OF HYDROXYLAMINE WITH THE WATER-OXIDIZING ENZYME INVESTIGATED VIA PROTON RELEASE*

Abstract— Photosynthetic water oxidation is a four-step redox process which is driven by a one-quantum-one-electron reaction center. Stepwise electron Abstract—ion from the water-oxidizing enzyme is accompanied by stepwise proton release with the following stoichiometric pattern at given half-rise times: 1 H⁺ (250 μs, S₀→ S₁):0 H⁺(S₁→ S₂): 1 H⁺ (200 μs, S₂→ S₃): 2 H⁺ (1.2 ms, S₃→ S₄→ S₀). (Förster and Junge, 1985, preceding article in this issue). Hydroxylamine at low concentrations (?100 μ M) appears to compete with water at the active site of the water- oxidizing enzyme. Its interference shifts the dark state of the water-oxidizing enzyme by two steps backwards (Bouges, 1971). We found that the hydroxylamine-induced shift was also reflected in the stoichiometric pattern and in the kinetics of proton-release. In the presence of hydroxylamine, two protons per reaction center were released with a half-rise time of ? 2 ms upon the first exciting flash given to dark adapted thylakoids. This was slower than observed for each of the protons released during unperturbed water oxidation. One proton was released upon the second flash. The half-rise time of the main component observed upon the second flash in hydroxylamine-treated samples agreed with the one observed upon the fourth flash in the absence of hydroxylamine, which had been attributed to the S₀→ S₁ transition. The two protons which were observed upon the first flash in hydroxylamine-treated thylakoids may be due to hydroxylamine oxidation or to the association of water to the catalytic manganese center after detachment of oxidized hydroxylamine from its binding site.     

BIPHASIC ENERGY CONVERSION KINETICS AND ABSORBANCE DIFFERENCE SPECTRA OF PHOTOSYSTEM II OF CHLOROPLASTS. EVIDENCE FOR TWO DIFFERENT PHOTOSYSTEM II REACTION CENTERS

Abstract— The continuous illumination induced kinetics of photochemical energy conversion at system II have been measured with isolated and 3-(3, 4-dichlorophenyl)-l, l-dimethylurea (DCMU) poisoned chloroplasts by means of absorbance difference spectroscopy in the UV and by the area growth over the fluorescence induction curve at room temperature. An optimal set of conditions was found in order to isolate absorbance changes caused by the reduction of the primary electron acceptor Q of PS II by suppressing other electron transfer processes. The light induced kinetics of Q- accumulation in the absorbance change measurements were found to be biphasic and strictly correlated with the kinetics of the area growth measured under the same conditions. From the resolution of the biphasic kinetics at different wavelengths in the UV region of the spectrum, it was found that both kinetic components in the system II photochemistry involve the reduction of a plastoquinone molecule to its plastosemiquinone anion. From the two kinetic components one was fast and non-exponential and the other relatively slow with an exponential time course. The initial rate difference in the kinetics of the two components was by a factor of approximately 3. A difference by a factor of about three was also found in the flash saturation curves of the two kinetic components.
The results are explained by the hypothesis that in higher plant chloroplasts there are system II reaction centers embedded in a large pigment matrix with statistical energy transfer, and system II reaction centers embedded in separate, in terms of excitation energy transfer, units. The effective absorption cross section per reaction center for the centers in the statistical pigment bed is approximately 3 times larger than that of the reaction centers in the separate system II units. The two types of system II reaction centers have different yields of excitation trapping and charge stabilization properties.     

Isolation of an Antiaromatic 9-Hydroxy Fluorenyl Cation

Fluorenyl cations are textbook examples of 4π electron antiaromatic five-membered ring systems. So far, they were reported only as short-lived intermediates generated under superacidic conditions or by flash photolysis. Attempts to prepare a m-terphenyl acylium cation by fluoride abstraction from a benzoyl fluoride gave rise to an isolable 9-hydroxy fluorenyl cation that formed by an intramolecular electrophilic attack at a flanking mesityl group prior to a 1,2-methyl shift and proton transfer to oxygen.     

Triplet State Energy Transfer Between the Primary Donor and the Carotenoid in Rhodobacter sphaeroides R-26.1 Reaction Centers Exchanged with Modified Bacteriochlorophyll Pigments and Reconstituted with Spheroidene

Abstract— The dynamics of triplet energy transfer between the primary donor and the carotenoid were measured on several photosynthetic bacterial reaction center preparations from Rhodobacter sphaeroides : (a) wild-type strain 2.4.1, (b) strain R-26.1, (c) strain R-26.1 exchanged with 13²-hy-droxy-[Zn]-bacteriochlorophyll at the accessory bacteriochlorophyll (BChl) sites and reconstituted with spheroidene and (d) strain R-26.1 exchanged with P-vinyl]-13²-hydroxy-bacteriochlorophyll at the accessory BChl sites and reconstituted with spheroidene. The rise and decay times of the primary donor and carotenoid triplet-triplet absorption signals were monitored in the visible wavelength region between 538 and 555 run as a function of temperature from 4 to 300 K. For the samples containing carotenoids, all of the decay times correspond well to the previously observed times for spheroidene (5 ± 2 us). The rise times of the carotenoid triplets were found in all cases to be biexponen-tial and comprised of a strongly temperature-dependent component and a temperature-independent component. From a comparison of the behavior of the carotenoid-con-taining samples with that from the reaction center of the carotenoidless mutant Rb. sphaeroides R-26.1, the temperature-independent component has been assigned to the buildup of the primary donor triplet state resulting from charge recombination in the reaction center. Arrhenius plots of the buildup of the carotenoid triplet states were used to determine the activation energies for triplet energy transfer from the primary donor to the carotenoid. A model for the process of triplet energy transfer that is consistent with the data suggests that the activation barrier is strongly dependent on the triplet state energy of the accessory BChl pigment, BChl_B.     

PHOTOSYNTHETIC OXYGEN EVOLUTION UNDER VARYING REDOX CONDITIONS: NEW EXPERIMENTAL AND THEORETICAL RESULTS

Abstract. –Steady state and transient effects of two electron transport inhibitors (NH₂OH˙HCl and DCMU) and one uncoupler (FCCP) upon the emission of delayed fluorescence have been studied in whole cells of Chlorella pyrenoidosa. Observations were made on changes in the delayed fluorescence emitted 0.5 ms after a brief flash and 60 ms after the extinction of steady illumination and on changes in the induction effects caused by preillumination. Patterns in the time course of events after inhibitor addition occur which have different characteristics for each of the additives. The observations are consistent with a model of delayed fluorescence production based on the recombination of charges.