A new site of bicarbonate effect in Photosystem II of photosynthesis: Evidence from chlorophyll fluorescence transients in spinach chloroplasts

Recent studies on oxygen evolution of corn chloroplast fragments in flashing light [Stemler, A., Babcock, G.T. and Govindjee (1974) Proc. Natl. Acad. Sci. 71, 4679–4683] have shown that the absence of bicarbonate ions increases the turnover time of the Photosystem II reaction center. The rate limiting steps in Photosystem II turnover can be interpreted in terms of reactions either on the oxidizing (electron donor) or reducing (electron acceptor) side of the reaction center. Experiments are reported here that suggest at least one site of bicarbonate action on the reducing side. In Triswashed spinach chloroplasts (incapable of O₂ evolution), the chlorophyll a fluorescence transient in the presence of various artificial electron donors (hydroquinone, diphenylcarbazide, MnCl₂ and NH₂OH) and in the absence of bicarbonate ions shows a rapid initial rise; the addition of 10 mM NaHCO₃ restores the transient to one characteristic of normal chloroplasts. Furthermore, the transients measured as a function of decreasing bicarbonate concentrations are qualitatively similar to those observed with increasing concentrations of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea which imposes a block on the reducing side, rather than to transients observed with increasing concentrations of NH₂OH or prolonged heat treatments, which impose a block on the oxidizing side.     

A flash photolysis ESR study of photosystem II signal IIvf, the physiological donor to P-680+.

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (g approximately 2.0045, deltaHpp approximately 19G). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with "red" illumination (lambda less than 680 nm) and occurs stoichiometrically in a 1:1 ratio with P-700+. The Photosystem II transient is formed in less than 100 mus and decays via first-order kinetics with a halftime of 400-900 mus. Additionally, the t1/2 for radical decay is temperature independent between 20 and 4 degrees C; however, below 4 degrees C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal-mol-1. Inhibition of electron transport through Photosystem II by o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1-5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this "quinoidal" type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, P-680+.     

The emission yields of delayed and prompt fluorescence from chloroplasts.

1. The decay of delayed fluorescence from chloroplasts blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and uncoupled with gramicidin has been measured in the time range 0.75--45 ms by use of a laser phosphoroscope. 2. The decays have been analysed as the sum of three first-order components of approximate half-lives 0.2, 2.5 and 300 ms by a computer-assisted least-squares fit procedure. 3. The prompt fluorescence yield of the chloroplasts was manipulated by changing the cation concentration of the chloroplast-suspending medium. 4. Analysis of the concentration dependence of the components of the delayed fluorescence decay and of the prompt fluorescence inductions indicates that the emission yield of the intermediate (tau approximately 2.5 ms) component of the decay is equal to the fluorescence yield of a Photosystem II photosynthetic unit with an open trap, and that for the slow (tau approximately 300 ms) component the emission yield is equal to the total Photosystem II prompt fluorescence yield. 5. It is concluded that the delayed fluorescence yield in the time range studied is a complex function of time, which may be due to there being different mechanisms leading to delayed fluorescence production at short and long times after cessation of illumination.     

Bacteriochlorophyll fluorescence of purple bacteria at low redox potentials. The relationship between reaction center triplet yield and the emission yield.

This work describes fluorescence yield measurements in suspensions of strains of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in which the iron . quinone complex (X) was chemically reduced (state [PIX-]; P is the reaction center bacteriochlorophyll dimer, I is the long wavelength bacteriopheophytin), and compares these with the fluorescence observed when all the traps are open (state [PIX]) and with the fluorescence observed when all the traps are closed (state [P+IX]). At 77 K the amplitude and the shape of the fluorescence emission spectrum in [PIX-] are identical to those observed in [PIX]. This is a strong indication that all the extra fluorescence observed at room temperature in [PIX-] is, in fact, caused by an efficient back reaction [P+I-X-] leads to [P*IX-]. Using an equation similar to the original Vredenberg-Duysens relationship (Vredenburg, W.J. and Duysens, L.N.M. (1963) Nature 197, 355-357) but now assuming that a single reaction center has a probability pt of trapping an excitation and (1--pt) of re-emitting it to the surroundings, we are able to calculate pt as a function of the temperature by measuring the fluorescence in [PIX], [PIX-] and [P+IX] as a function of the temperature. The calculated pt values agree reasonably well with triplet yields measured in isolated reaction centers. Finally, we have measured the reaction center triplet yield (PTR) in intact systems and we have shown that the sum of the triplet yield and the remaining loss processes (PL) in the antenna bacteriochlorophyll including the bacteriochlorophyll dimer (such as fluorescence, internal conversion or direct triplet formation) is approximately constant; if we assume that at 77 K the only process which occurs in the reaction center is the formation of a reaction center triplet, than PTR + PL=1. The energy barrier between [P*IX-] and [P+I-X-] was estimated to be 0.11--0.15 eV for a set of preparations.     

A slow component of delayed light emission as a function of temperature mimics glow peaks in photosynthetic membranes. Evidence for identity

Evidence for a correlation between a slow component of delayed light emission and thermoluminescence from photosynthetic membranes is presented. It was observed that the intensity of delayed light measured 2.5 s subsequent to illumination at different temperatures when plotted as a function of temperature reproduces the glow curve pattern. The slow component of delayed light emission is also quantitatively related to the yield of thermoluminescence, the sum of the two remaining constant.     

The effect of detergent Triton X=100 on the light induced changes in the fluorescence yield of chloroplasts]

It is shown that light induced changes of the fluorescence yield (delta F) of isolated chloroplasts are affected by Triton X-100. delta F value descreases with the increase of the detergent concentration from 0 to 0.03%, increases in the range of 0.03--0.05% and is irreversibly blocked at concentrations more than 0.08--0.1%. The same dependence of delta F on the detergent concentration is obtained for "digitonin" fragments of chloroplasts enriched in the photosystem 2, but not for fragments enriched in the photosystem 1. Light induced delta F of chloroplasts treated by detergent were activated by hydroxylamine and saturated at lower light intensities than delta F of untreated chloroplasts. Addition of 0.01% Triton resulted in an activation of light induced delta F of chloroplasts with damaged donor part of photosystem 2. It is suggested that the complex dependence of delta F of chloroplasts on the Triton concentration is due to superposition of several effects: the uncoupling of photophosphorylation, inactivation of the electron transport chain in the donor and acceptor parts of photosystem 2, and changes of acting concentration of Triton X-100 within the range of critical micelle concentration.     

Studies on the relation between the fast phase of millisecond delayed light emission and the proton released from oxidation of water in spinach chloroplast

The relation between the fast phase of ms-DLE (delayed light emission measured with a phosphoroscope) and the proton released from water oxidation in spinach chloroplasts was studied in several aspects. When photophosphorylation was allowed to be coupled to the Hill reaction the intensity of the fast phase of ms-DLE of chloroplast was lowered more at 1 °C than at 25 °C, and the photophosphorylation rate within 40 ms of flashing light was higher at 1 °C than at 25 °C. Adding the subunit of ATP synthase to the chloroplast preparation to block the leakage of protons through ATP synthase, the intensity of the fast phase of ms-DLE was enhanced, to a larger extent at 1 °C than at 25°C. When the ms-DLE was measured under isotonic conditions, the intensity of fast phase of ms-DLE enhanced by proton released from oxidation of water was more pronounced. The above results support the suggestion that under lower temperature and isotonic conditions, the proton released from water oxidation was liable to be localized and could enhance the intensity of the fast phase of ms-DLE more effectively.     

Transients of delayed fluorescence induction signal and photosynthetic antennas: A possible relationship. Mathematical modeling approach

A mathematical model was developed for resolved temporal transients of experimentally recorded delayed fluorescence (DF) induction signal. During an intermittent light regime, antennas of the photosynthetic apparatus were treated as targets, repeatedly hit by potentially absorbable photons within a series of consecutive light flashes. Formulas were derived for the number of antennas, cumulatively hit by a specific number of photons, as a function of the flash serial number (time). Model parameters included number of absorbable photons in one flash, antenna sizes, and their number. A series of induction curves were analyzed, obtained from a Zea mays leaf segment and differing in the previous dark period (t _d). Each curve, consisting of the two most prominent DF transients (C and D), was fitted with several model types, differing in the number of absorbed photons. For both transients, the best fitting result was achieved when DF induction was linked to the second absorbed photon. As expected, model parameters related to antenna sizes showed weaker dependence on t _d than those referring to antenna number. With restrictions applied to this model, the two DF induction transients may be related to two classes of photosynthetic antennas. Their different sizes may have a predominant influence on the efficiency of photon absorption and possibly time-dependent appearance of DF transients. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 325–335. The text was submitted by the authors in English.     

The major antenna complex of photosystem II has a xanthophyll binding site not involved in light harvesting.

We have characterized a xanthophyll binding site, called V1, in the major light harvesting complex of photosystem II, distinct from the three tightly binding sites previously described as L1, L2, and N1. Xanthophyll binding to the V1 site can be preserved upon solubilization of the chloroplast membranes with the mild detergent dodecyl-alpha-d-maltoside, while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2, and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesized zeaxanthin, which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.     

Action of salicylaldoxime on electron transport reactions,fluorescence yield,and light-induced field changes in spinach chloroplasts: A new mode of inhibition in photosystem II

Salicylaldoxime (1–10 mm) inhibits chloroplast electron transport reactions by a reversible and an irreversible modification of photosystem II. The irreversible inhibition correlates with removal of the loosely bound pool of manganese associated with the water-splitting mechanism. The reversible inhibition is characterized by (i) a suppression of artificial donor reactions, (ii) a high initial fluorescence yield, and (iii) a decline in the amplitude of the flash-induced electric field across the membrane. After removal of the inhibitor, the initial fluorescence yield declines to near-control levels, but the variable portion of the fluorescence rise remains missing. Addition of an artificial donor restores the variable fluorescence yield and normal electron transport rates to 2,6-dichlorophenolindophenol. Characteristics of the reversible inhibition suggest that salicylaldoxime causes suppression of photochemical charge separation in photosystem II.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Thermally-induced delayed fluorescence of photosystem I and II chlorophyll in thermophilic cyanobacterium Synechococcus elongatus.

Stationary delayed fluorescence (DF) of chlorophyll in isolated membrane preparations from thermophilic cyanobacterium Synechococcus elongatus was investigated as a function of temperature. Two peaks at different temperatures were observed. The low-temperature peak (54-60 degrees C) coincided with the main maximum of the thermally-induced delayed fluorescence of chlorophyll in intact cells and PSII-particles with active oxygen-evolving system. The high-temperature peak (78 degrees C) coincided with the minor band of delayed light emitted by intact cells. It was also observed in the delayed fluorescence emission from a PSI-enriched fraction preparation. The intensities of the DF peaks were dependent on the presence of inhibitors, donors and acceptors that cause specific effects on electron transport of the two photosystems. The low-temperature and high-temperature peaks were related to PSII and PSI, respectively. The manifestation of delayed fluorescence from PSI and PSII at different temperatures seems to be a specific property of thermophilic cyanobacteria. The reason for this may be a high thermal stability of the photosystems and the lack of the PSII antenna complex in isolated membranes. Consequently, the relative yield of delayed fluorescence from PSI markedly increases. Thermally-induced fluorescence seen in membranes of cyanobacteria showed a high sensitivity to structural and functional membrane alterations induced by pH changes, different electron transport stabilizing agents or different concentrations of MgCl2.     

Cation control of fluorescence emission,light scatter,and membrane stacking in pigment mutants of Chlamydomonas reinhardi

The effect of monovalent and divalent cations on thylakoid membrane stacking, light scatter, and fluorescence yield were examined in broken-cell preparations of the wild type of Chlamydomonas reinhardi and mutants lacking various pigment-protein complexes. Membrane stacking as determined by electron microscopy and light scatter at 540 nm shows an approximate linear proportionality. In a mutant lacking photosystem II reaction centers, stimulation of light scatter and of fluorescence yield show different kinetics at a given cation concentration and show different titration curves as a function of cation concentration. Control of membrane stacking and fluorescence yield is attributed, respectively, to two locations differing in their anionic charge density. In a mutant lacking chlorophyll-protein complex I, the cation effect on the fluorescence yield is reversed giving rise to a decrease in fluorescence intensity upon cation addition instead of the usual increase. We conclude that the site of energy spillover between the two photosystems is located exterior to chlorophyll-protein complex I but not at the junction of chlorophyll-protein complex I and the rest of the light-harvesting antenna.