Flash-induced Absorption Changes Research Articles

Direct observation of the immediate electron donor to chlorophyll- a+II (P-680 +) in oxygen-evolving photosystem II complexes Resolution of nanosecond kinetics in the UV

Flash-induced absorption changes in the UV have been monitored with nanosecond time resolution in oxygen-evolving photosystem II complexes from Synechococcus sp. At 260 nm, the oxidation of the immediate donor to Chl- a + II (P-680 +) in the 10–500 ns range could be observed directly. The difference extinction coefficient for its oxidation is estimated to be 6000–8000 M −1·cm −1 at 260 nm. The observed kinetics for the oxidation of the donor is identical with the reduction kinetics of Chl- a + II as monitored at 824 nm. At 320 nm, the reduction of Q A was found to be faster than 1 ns.

Magnetic-field effects on primary reactions in Photosystem I

Magnetic-field effects on primary reactions in Photosystem I have been studied by measuring flash-induced absorption changes at 820 nm in SDS-CP1 particles in magnetic fields up to 1000 G. The amplitude of a transient decay phase with t 1 2 ≈ 6 μ s , which is attributed to the triplet state of P-700 formed by charge recombination in the primary radical pair (P-700 +-A − 0), shows a clear increase with increasing field (maximum effect at around 60 G), followed by a decrease to a level well below the control without field. The magnetic-field dependence of the triplet yield is discussed with regard to magnetic interactions between P-700 + and A − 0: an exchange interaction |2 J| of approx. 60 G is deduced (in accordance with a previous estimation of 60–100 G: Voznyak, V.M., Ganago, I.B., Moskalenko, A.A. and Elfimov, E.I. (1980) Biochim. Biophys. Acta 592, 364–368).

Excitation transfer in chlorosomes of green photosynthetic bacteria

The formation of excited states and energy transfer in chlorosomes of the green photosynthetic bacteria Chlorobium limicola and Chloroflexus aurantiacus were studied by measurements of flash-induced absorbance changes and fluorescence. Upon excitation with 35 ps, 532 nm flashes, large absorbance decreases around 750 nm were observed that were due to the disappearance of ground state absorption of the main pigment, bacteriochlorophyll (BChl) c. The absorbance changes decayed after the flash with a time constant of approx. 1 ns, together with faster components. Absorbance changes that could be ascribed to formation of excited BChl a were much smaller than those of BChl c. The yields of BChl c and BChl a fluorescence were measured as a function of the energy density of the exciting flash. At high energy a strong quenching occurred caused by annihilation of singlet excited states. An analysis of the results shows that energy transfer between BChl c molecules is very efficient and that in C. limicola excitations can probably move freely through the entire chlorosome (which contains about 10 000 BChls c). The chlorosome thus serves as a common antenna for several reaction centres. The small amounts of BChl a present in the chlorosomes of both species form clusters of only a few molecules. Upon cooling to 4 K the sizes of the domains of BChl c for energy transfer decreased considerably. The results are discussed in relation to recently suggested models for the pigment organization within chlorosomes.

Interaction of chlorophyll a′ with the 65 kDa subunit protein of photosystem I reaction center

The 65 kDa polypeptide subunit depleted of P700 was prepared from a photosystem I reaction center preparation and mixed with chlorophyll a′ (C-10 epimer of chlorophyll a) to yield a complex exhibiting a tripleheaded spectrum with absorbance maxima at 673, 692 and 707 nm. The difference spectra (oxidized-minus-untreated and light-minus-dark) had a major trough at 707 nm and minor ones at 690 and 430 nm. The overall shape of the spectra resembled well that of P700 with a small red shift. A rapidly decaying flash-induced absorbance change was observed at 430 nm with a half decay time of less than 500 μs in a preparation supplemented with an electron donor system.

The primary reaction of photosystem II in the D1-D2-cytochrome b-559 complex

Flash-induced absorbance changes in the picosecond and nanosecond time range have been measured in the ‘D1-D2-cyt b-559 complex’ isolated from photosystem II membranes. The results indicate the efficient formation of the primary radical pair P680 + pheophytin −, which had a lifetime of about 36 ns, and the presence of unconnected chlorophyll in this preparation. It is concluded that the complex contains the active photosystem II reaction center, and that this reaction center contains at most 4 chlorophyll a molecules.

Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis

The initial oxidized species in the photochemical charge separation in reaction centers from Rps. viridis is the primary donor, P(+), a bacteriochlorophyll dimer. Bound c-type cytochromes, two high potential (Cyt c 558) and two low potential (Cyt c 553), act as secondary electron donors to P(+). Flash induced absorption changes were measured at moderate redox potential, when the high potential cytochromes were chemically reduced. A fast absorption change was due to the initial oxidation of one of the Cyt c 558 by P(+) with a rate of 3.7×10(6)s(-1) (τ=270nsec). A slower absorption change was attributable to a transfer, or sharing, of the remaining electron from one high potential heme to the other, with a rate of 2.8×10(5)s(-1) (τ=3.5 μsec). The slow change was measured at a number of wavelengths throughout the visible and near infrared and revealed that the two high potential cytochromes have slightly different differential absorption spectra, with α-band maxima at 559 nm (Cyt c 559) and 556.5 nm (Cyt c 556), and dissimilar electrochromic effects on nearby pigments. The sequence of electron transfers, following a flash, is: Cyt c 556→Cyt c 559→P(+). At lower redox potentials, a low midpoint potential cytochrome, Cyt c 553, is preferentially oxidized by P(+) with a rate of 7×10(6)s(-1) (τ=140 nsec). The assignment of the low and high potential cytochromes to the four, linearly arranged hemes of the reaction center is discussed. It is concluded that the closest heme to P must be the high potential Cyt c 559, and it is suggested that a likely arrangement for the four hemes is: c 553 c 556 c 553 c 559P.

Stoichiometries and energetics of proton translocation coupled to electron transport in chloroplasts

We have investigated the stoichiometry of electron transport-driven proton uptake from the external medium and proton release into thylakoid vesicles of chloroplasts during single-turnover flash illumination. Proton uptake from the external medium was measured using a glass pH-electrode. For each electron originating from the oxidation of water by Photosystem II and ultimately forming H2O2 via the photoreduction of methyl viologen, approx. 2.7 protons were taken up from the external medium. For each electron originating from the oxidation of duroquinol by the cytochrome bf complex and ultimately forming H2O2 via methyl viologen under conditions in which water oxidation was inhibited by diuron, approx. 1.7 protons were taken up from the external medium. It is presumed that for each electron involved in the formation of H2O2 that one proton is taken up. Therefore, these results indicate that the electron-transfer reaction from H2O to Photosystem I involves the uptake of approx. 1.7 protons per electron, and that in the reaction from duroquinol to Photosystem I approx. 0.7 protons per electron are taken up. Proton release within the thylakoid vesicles was measured spectrophotometrically using the pH-sensitive dye neutral red. Flash-induced absorbance changes due to neutral red indicated that for each proton released within the thylakoid vesicle due to water oxidation approx. 1.9 protons appear inside the vesicle as a result of quinol oxidation. The flash-induced formation of ATP was determined by the incorporation of 32Pi into ATP. The initiation of flash-induced ATP formation by Photosystem II operating alone required nearly twice as many flashes as were necessary when the Photosystem I-dependent turnover of the cytochrome bf complex was responsible for proton accumulation, and nearly three times more than when Photosystem II and the cytochrome bf complex operated together. This is taken to indicate that both of the reactions that result in proton accumulation within the vesicle contribute energetically to ATP synthesis and contribute in proportion to their measured H+e− ratio. All of these flash-induced measurements, proton uptake from the external medium, proton release inside the vesicle, and the filling of the energetic threshold pool required for the onset of ATP synthesis, demonstrate that for each electron transferred from water through the cytochrome bf complex to Photosystem I nearly three protons are accumulated within the thylakoid membrane. Thus, these data indicate that oxidation of quinol by the cytochrome bf complex is able to support the translocation of two protons per electron during flash energization.

Dependence of energization of thylakoids on frequency of exciting flashes in intact chloroplasts

We investigated the frequency-dependence of the flash-induced electrochromic absorbance change, ΔA515, and of the pH-indicating absorbance change of neutral red in isolated intact chloroplasts. The energization pattern of thylakoids depended strongly on the frequency (f) of the exciting flashes, tested between 0.05 and 2 s(-1). When the frequency was increased from 0.1 to 1 s(-1) the total initial change and the slow rise of ΔA515 decreased by about 30% and 70%, respectively, and both the slow rise and decay were considerably accelerated. These changes were fully reversible, even after prolonged excitation at 1 s(-1), if the frequency was decreased again to 0.1 s(-1). Accumulation of an appreciable transmembrane electric field strength could not be detected in any of our experiments, at high frequency, since the decay of ΔA515 was considerably accelerated when the frequency was increased. In contrast, ΔpH significantly increased at higher frequencies of the exciting flashes. In the steady-state (after about 100 flashes) ΔpH was about 0.5-0.8 pH unit higher than in the dark or at low frequencies. In the presence of nigericin or dithionite, both of which prevented accumulation of protons in the lumen, the total initial change in ΔA515 at f=1 s(-1) relative to that at f=0.1 s(-1) decreased to a similar extent as in the control. The proportion of the slow rise relative to the initial amplitude, however, did not decrease. Our data support the suggestion that ΔpH controls the amplitude of the slow rise of ΔA515. However, contrary to a previous statement (B. Bouges-Bouquet (1981) Biochim. Biophys. Acta 535, 327-340), we show that the ΔpH effect cannot be accounted for by variation of the rate of this kinetic component of ΔA515.

A new intermediate in the photocycle of bacteriorhodopsin

With increasing pH (6.0 to 10.5) there is an increasing discrepancy between the recovery rate of bacteriorhodopsin after a flash and the decay rates of the key photointermediates, M fast and M slow, which is not predicted by any model of the photocycle. However, a very slowly decaying absorbance change at 350 nm has kinetics which match the very slow part of the recovery of bacteriorhodopsin, suggesting a new intermediate. The difference spectra of the bacteriorhodopsin photocycle measured at pH 10.5, obtained from fitting 3 exponentials to the flash-induced absorbance changes in the 300–560 nm wavelength range, show maxima, at 410–420 nm corresponding to the 2 forms of M and at 350 nm corresponding to the new intermediate. We propose to call this new species R350.

Genetically engineered mutant of the cyanobacterium Synechocystis 6803 lacks the photosystem II chlorophyll-binding protein CP-47

CP-47 is absent in a genetically engineered mutant of cyanobacterium Synechocystis 6803, in which the psbB gene [encoding the chlorophyll-binding photosystem II (PSII) protein CP-47] was interrupted. Another chlorophyll-binding PSII protein, CP-43, is present in the mutant, and functionally inactive PSII-enriched particles can be isolated from mutant thylakoids. We interpret these data as indicating that the PSII core complex of the mutant still assembles in the absence of CP-47. The mutant lacks a 77 K fluorescence emission maximum at 695 nm, suggesting that the PSII reaction center is not functional. The absence of primary photochemistry was indicated by EPR and optical measurements: no chlorophyll triplet originating from charge recombination between P680(+) and Pheo(-) was observed in the mutant, and there were no flash-induced absorption changes at 820 nm attributable to chlorophyll P680 oxidation. These observations lead us to conclude that CP-47 plays an essential role in the activity of the PSII reaction center.

Effects of arginine modification on the photocycle of halorhodopsin

Exhaustive reaction with phenylglyoxal removed 9 of the 12 arginine and 1 of the 2 lysine residues in detergent-solubilized halorhodopsin, without affecting the chromophore. The consequences of this extensive removal of positive charges on various chloride-binding equilibria and the photochemistry were evaluated. No significant effects were seen on the affinity of Site I to chloride and on the increase in the p K a of Schiff-base deprotonation, which is caused by the chloride binding at this site. No significant effects were seen on the affinity of Site II to chloride, either. However, the photocycle of the pigment was affected. Kinetic modeling of the observed changes in flash-induced absorption changes suggests that the modification increases the affinity of the main halorhodopsin photointermediate to chloride by about fourfold. If chloride translocation involves release of chloride from this intermediate during the transport cycle, the result might explain the observed partial inhibitory effects on chloride transport. Plausible models of chloride translocation include reversible binding of the anion by positively charged groups, strategically arranged in the protein. The results indicate that two of the three spectroscopically observable chloride-dependent equilibria do not depend on a large number of positively charged residues in the protein. To the extent that the unaffected equilibria represent association and dissociation which occur during chloride translocation, at least part of the chloride translocation might be accomplished with the participation of only a few positively charged residues.

Flash-induced absorption changes in photosystem I at low temperature: evidence that the electron acceptor A 1 is vitamin K 1

Low temperature flash absorption spectroscopy has been applied to elucidate the chemical nature of the secondary electron acceptor A 1 of photosystem I (PS-I). The flash-induced absorption changes measured in digitonin-fractionated spinach PS-I particles at 10 K between 240 and 525 nm are shown to comprise a major decay phase with t sol1 2 ~ 150 μs which has been attributed to the recombination reaction P-700 +·A 1 → P-700·A 1 [(1984) Biochim. Biophys. Acta 767, 404-414]. We present the absorption difference spectrum of this reaction and demonstrate that it contains contributions in the ultraviolet due to A 1, which are characteristic of vitamin K 1 (phylloquinone).

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q ⨪ B) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential ( E h) to 200–300 mV is the result of increasing the lifetime of Q ⨪ B, as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q ⨪ B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q ⨪ B, the flash-induced redox changes of Q ⨪ B were observed at low E h values unless E h reached a value at which Q B underwent reduction at equilibrium to form Q BH 2. The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q BH 2) of the secondary acceptor.

Electrical potential changes in the surface and the central region of chromatophore membranes of photosynthetic bacteria detected by the absorbance changes of ethidium and carotenoid

(1) Changes of local intramembrane electrical field in the surface and central region of the chromatophore membrane during energization were studied both by the measurement of absorbance changes of ethidium, a monovalent cationic dye, and of carotenoid, the intrinsic probe of electrical field. (2) Binding of ethidium to the chromatophore membrane of Rhodopseudomonas sphaeroides was found to be dependent on the energization of membrane as well as on the ionic condition of the medium. The dye was released from the membrane when salt was added to the suspension, indicating the electrostatic interaction between the positive dye and the net negative membrane surface. The result was explained by the surface-potential dependent distribution of the dye to the membrane surface, as seen with other charged dyes (Masamoto, K., Matsuura, K., Itoh, S. and Nishimura, M. (1981) Biophys. Acta 638, 108–115). (3) Energization of chromatophores by flash-light-induced absorbance change of ethidium showing a similar difference spectrum to that induced by the addition of salts. The release of ethidium by a single turn-over flash of saturating intensity was estimated to be 0.22 ethidium per reaction center. Addition of ethidium (at 200 μM) slightly affected the flash-induced absorbance change of carotenoid which responds to the intramembrane electricalfield change, indicating a low-membrane permeability of the dye. The extent of the absorbance change of ethidium was linear to that of carotenoid, and was abolished in the presence of valinomycin plus K +. However, the rise and decay kinetics of the absorbance change of ethidium was different from that of carotenoid. (4) These absorbance changes of ethidium and carotenoid can be explained by a model in which ethidium responds to the potential changes in the surface region and carotenoid in the central hydrophobic region of the chromatophore membrane.

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. II. Higher plant leaves

The flash-induced electrochromic absorbance change (ΔA515) was measured in leaves of higher plants in the absence and presence of continuous monochromatic background illumination of different intensities and wavelengths. The variation of the amplitude of ΔA515 in background light was used to estimate the steady-state turnover time of the electron transport. In red light we obtained about 5 msec which was accounted for by the turnover of the linear electron transport. With far red background illumination or in the presence of the photosystem 2 inhibitor, DCMU, the steady-state turnover time tentatively assigned to photosystem 1 cyclic electron transport was much larger (≥100 msec).

PH and salt effects on the slow intermediates of the bacteriorhodopsin photocycle

Purple membrane fragments of Halobacterium halobium were used to investigate pH and salt effects on the kinetics of M412, O660 and BR568. The flash-induced absorbance changes were measured in the 5–9 pH range, at low ionic strength and at 4 M NaCl. The results are consistent with a model which implies a branching in the last part of the bacteriorhodopsin photocycle.

Electron transport from cytochrome b6- f complexes to Photosystem I reaction center complexes in Synechococcus sp. Is cytochrome c-553 a mobile electron carrier?

Numbers of the Photosystem I reaction center complexes and the cytochrome b 6- f complexes with which a cytochrome c-553 molecule can interact within the limiting time of photosynthetic electron transport were examined by measuring flash-induced absorption changes of P-700, cytochrome c-553 and cytochrome f in the thermophilic cyanobacterium Synechococcus sp. The addition of 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB) did not affect the common 2 ms half-time of P-700, cytochrome c-553 and cytochrome f reduction, which is ascribed to electron transfer from the plastoquinone pool. The inhibitor decreased, however, amounts of the three electron carriers which underwent the 2 ms reduction in the order of cytochrome f, cytochrome c-553 and P-700. On excitation with weak flashes which oxidized only a small fraction of cytochrome c-553 molecules present in cells, P-700 remained in the oxidized state after the flashes was reduced with electrons from the Rieske center or plastoquinone but not from cytochrome c-553. The ratios of cytochrome c-553 to cytochrome f oxidized at various flash intensities were constant and similar to the ratio of the two cytochromes present in cells. It is concluded that cytochrome c-553 cannot exchange electrons with large numbers of the Photosystem I reaction center complexes and the cytochrome b 6- f complexes in the limiting time, but has a mobility sufficient to mediate electron transfer between the two complexes, which are present at an unbalanced ratio in Synechococcus cells.

Effects of herbicides on binary oscillations of ultraviolet flash-induced absorption changes in chloroplasts

The competition between various herbicides and plastoquinone, at the Q B site of Photosystem II, has been studied by measuring absorption changes between 300 and 360 nm in spinach and chenopod chloroplasts, in response to a train of saturating short xenon flashes. A complex pattern was observable without addition of chemicals interfering with electron flow. The effect of potassium ferricyanide, of hydroxylamine and of valinomycine-K + permitted to simplify the pattern and, in particular, to observe binary oscillations with flash number, attributable to the functioning of the two-electron gate Q B. Herbicides belonging to different classes block electron transfer when added at high concentration. At low concentration, however, inverted binary oscillations become observable. When the chloroplasts have been first oxidized with ferricyanide, this behaviour develops progressively in response to illumination. Varying the herbicide concentration, it appears that the concentration inducing maximum binary oscillations correlates with inhibition of linear electron transfer, within each class of herbicides. Phenolic herbicides induce the largest oscillations and ureas the smallest. The binary oscillations have the spectrum of the plastoquinone anion. The results clearly show that the studied herbicides compete efficiently with Q B, but not with Q − B, at the Q B binding site. Atrazine-resistant chenopod chloroplasts still display normal binary oscillations in the absence of herbicide, or in the presence of atrazine alone. They are highly sensitive to DCMU and to i-dinoseb, but no inverted binary oscillations could be observed with these herbicides.

Redox study of electron donation to P-680 in Photosystem II

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680 + in both preparations showed biphasic recovery phases with half-times of 42 and 625 μs at pH 4.5. The magnitude of the 42 μs phase of P-680 + rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D 1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 ± 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5–6.0. In contrast, the magnitude of the 625 μs phase of P-680 + rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with E m ≈ 240 mV, termed D 0, serves as a rapid donor ( t 1 2 ≤ 2 μ s ) to P-680 + in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D 1, is functional only when D 0 becomes oxidized.

Kinetics of manganese redox transitions in the oxygen-evolving apparatus of photosynthesis

The kinetics of the S-state transitions of the oxygen-evolving complex were analyzed in dark-adapted, oxygen-evolving Photosystem-II preparations supplied with the electron acceptor 2,5-dichloro- p-benzoquinone. The kinetics of flash-induced absorbance changes at 350 nm, largely due to the successive S-state transitions S 0 → S 1, S 1 → S 2, S 2 → S 3 and S 3 →; S 0, confirm the +1, +1, +1, −3 sequence of manganese oxidation reported earlier (Dekker, J.P., Van Gorkom, H.J., Wensink, J. and Ouwehand, L. (1984) Biochim. Biophys. Acta 767, 1–9), and reveal half-times of 30, 110, 350 and 1300 μs, respectively, for these transitions.