Electron transfer and proton pumping under excitation of dark-adapted chloroplasts with flashes of light

Proton release inside thylakoids, which is linked to the action of the water-oxidizing enzyme system, was investigated spectrophotometrically with the dye neutral red under conditions when the external phase was buffered. Under excitation of dark-adapted chloroplasts with four short laser flashes in series, the pattern of proton release as a function of the flash number was recorded and interpreted in the light of the generally accepted scheme for consecutive transitions of the water-oxidizing enzyme system: S₀ → S₁, S₁ → S₂, S₂ → S₃, S₃ → S₄, S₀. It was found that the proton yield after the first flash varied in a reproducible manner, being dependent upon the dark pretreatment given. In terms of the proton-electron reaction during these transitions, the pattern was as follows. In strictly dark-adapted chloroplasts (frozen chloroplasts thawed in darkness and kept for at least 7 min in the dark after dilution), it was fitted well by a stoichiometry of 1:0:1:2. In a less stringently dark-adapted preparation (as above but thawed under light), it was fitted by 0:1:1:2. Mechanistically this is not yet understood. However, it is a first step towards resolving controversy over this pattern among different laboratories. Under conditions where the 1:0:1:2 stoichiometry was observed, proton release was time resolved. Components with half-rise times of 500 and 1000 μs could be correlated with the S₂ → S₃ and S₃ → S₄ transitions, respectively. Proton release during the S₀ → S₁ transition is more rapid, but is less well attributable to the transitions due to error proliferation. A distinct component with a half-rise time of only 100 μs was observed after the second flash. Since it did not fit into the expected kinetics (based on literature data) for the S_i → S_i+1 transitions, we propose that it reflects proton release from a site which is closer to the reaction center of Photosystem (PS) II than the water-splitting enzyme system. This is supported by the observation of rapid proton release under conditions where water oxidation is blocked. Related experiments on the pattern of proton uptake at the reducing side of PS II indicated that protons act as specific counterions for semiquinone anions without binding to them.     

The rate of Qx→Qy relaxation in bacteriochlorophylls of reaction centers from Rhodobacter sphaeroides determined by kinetics of the ultrafast carotenoid bandshift

Transient absorption changes induced by excitation of isolated reaction centers (RCs) from Rhodobacter sphaeroides with 600 nm laser pulses of 20 fs (full width at half maximum) were monitored in the wavelength region of 420–560 nm. The spectral features of the spectrum obtained are characteristic for an electrochromic band shift of the single carotenoid (Car) molecule spheroidene, which is an integral constituent of these RCs. This effect is assigned to an electrochromic bandshift of Car due to the local electric field of the dipole moment formed by electronic excitation of bacteriochlorophyll (BChl) molecule(s) in the neighborhood of Car. Based on the known distances between the pigments, the monomeric BChl (B_B) in the inactive B-branch is inferred to dominate this effect. The excitation of B_B at 600 nm leads to a transition into the S₂ state (Q_x band), which is followed by rapid internal conversion to the S₁ state (Q_y band), thus leading to a change of strength and orientation of the dipole moment, i.e., of the electric field acting on the Car molecule. Therefore, the time course of the electrochromic bandshift reflects the rate of the internal conversion from S₂ to S₁ of B_B. The evaluation of the kinetics leads to a value of 30 fs for this relaxation process. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

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 S₀ → S₁, S₁ → S₂, S₂ → S₃ and S₃ →; S₀, 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.     

What can we still learn from the electrochromic band-shifts in Photosystem II?

Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the Q_X absorption of Phe_D1 differs, a band-shift in the Q_X region of Phe_D2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of Tyr_D. In contrast, a band-shift due to the formation of either Q_A^•- or Tyr_Z^• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to Phe_D1 and at ~ 544 nm as expected with Q130 H-bonded to Phe_D1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O₂-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of P_D1 is blue shifted and in the PsbA3/T179H mutant. Upon Tyr_Z^•Q_A^•- formation the Soret band of P_D1 is red shifted and the Soret band of Chl_D1 is blue shifted. In contrast, only P_D1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to P_D1 is biphasic for all the S-state transitions except for S₁ to S₂, and shows that: i) the proton release in S₀ to S₁ occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S₂ to S₃, in T. elongatus, are significantly faster than often considered. The nature of S₂Tyr_Z^• is discussed in view of the models in the literature involving intermediate states in the S₂ to S₃ transition.     

Optical characterization of intermediates in the water-splitting enzyme system of photosynthesis — possible states and configurations of manganese and water

Absorption changes coupled with the individual transitions S₀–S₃ and redox reactions in the water-splitting enzyme system S of photosynthesis have been measured. The principal difficulties of measuring the very small absorption changes in the ultraviolet coupled with those reactions have been reduced drastically through the use of a highly purified Photosystem II complex isolated from the Cyanobacterium synechococcus. The general problem caused by the mixing of the S states during a train of flashes and the falsification through the overlap with absorption changes of Q_B (binary oscillations) have been treated as follows. (1) The binary oscillations were bypassed through the use of silicomolybdate and high concentrations of DCBQ, respectively, as external electron acceptor. (2) Stable absorption changes of the mixed S-state transitions have been deconvoluted through fitting procedures to get the changes of the individual transitions of S₁ → S₂ → S₃ → S₀ → S₁. (3) Kinetically resolved absorption changes of the S-states in the 100-μs range gave independent information on the individual transitions. (4) Stable absorption changes of the S₀ → S₁ transitions in the forefront were induced after shifting the S states through low concentrations of NH₂OH two units backwards. Analysis of the resulting sequence S_x → S₀ → S₁ → S₂ → S₃ → S₀, beginning with an NH₂OH depending pre-state, S_x, and followed by an S₀ → S₁ transition not mixed with the opposite S₃ → S₀ transition, increased the conclusiveness considerably. It results that the ultraviolet spectrum of the S₀ → S₁ transition is different from the spectra of the S₁ → S₂ and S₂ → S₃ transition. Possible states of manganese, water and surplus charges responsible for these spectra are presented.     

Two-photon excitation spectrum of fluorescence of the light-harvesting complex B800–850 from Allochromatium minutissimum within 1200–1500 (600–750) nm spectral range is not carotenoid mediated

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ～ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S₀ → S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.     

S-state turnover in the O2-evolving system of CaCl2-washed Photosystem II particles depleted of three peripheral proteins as measured by thermoluminescence. Removal of 33 kDa protein inhibits S3 to S4 transition

The S-state transition in manganese-containing spinach PS II particles depleted of three peripheral proteins (33, 24 and 16 kDa) by CaCl₂ washing was investigated by means of thermoluminescence measurements and the following results were obtained: (1) When excited by continuous light, these particles showed the same glow curves as those of control PS II particles having a marked peak around +35°C (B band) which arises from recombination between S₂ (or S₃), the oxidized species of the so-called S states of the water-oxidation enzyme, and Q⁻_B, the semiquinone form of the secondary plastoquinone acceptor of PS II. (2) When excited, however, by a series of flashes, oscillation of the B band in the depleted particles proceeded normally up to the 2nd flash but was interrupted thereafter; in contrast, the control particles underwent quadruple oscillation showing maxima at the 1st and 5th flashes. (3) The oscillation pattern of the depleted particles agreed well with a computer simulation pattern obtained by assuming inhibition of the S₃ → S₄ transition. (4) The B-band height created by one or two flashes in the depleted particles showed decay kinetics almost the same as those in control particles. (5) During incubation in a low-salt medium, the B-band height of the depleted particles gradually decreased concomitant with release of manganese from the particles, and reached a zero level when about half of the manganese atoms were lost. (6) Removal of the 24 and 16 kDa proteins by NaCl washing appreciably lowered the B-band height, but did not affect at all the oscillation pattern of the B-band. These results indicate that the manganese catalyst in CaCl₂-washed PS II particles is unable to undergo the S₃ → S₄ transition because of depletion of the 33 kDa protein, while the catalyst is still capable of undergoing S₀ → S₁, S₁ → S₂ and S₂ → S₃ transitions.     

Cooperative and reversible action of three or four hydroxylamine molecules on the water-oxidizing complex

Photosynthetic water oxidation is a four-step oxidation process which is formally described by the transitions S₀ → S₁, S₁ → S₂, S₂ → S₃, S₃ → S₄ → S₀. In dark-adapted thylakoids S₁ is the most stable and oxygen is liberated after abstraction of three oxidizing equivalents from the water-oxidizing complex. If hydroxylamine is present at low concentrations (15–100 μM), five instead of three oxidizing equivalents have to be abstracted before dioxygen is liberated. We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. We obtained an apparent Hill coefficient of 2.43. This result could be explained by cooperative binding of 3 or 4 hydroxylamine molecules, and by the assumption that each center which was modified by >= 1 hydroxylamine molecule gave rise to the same modified pattern of proton release.     

Energy transfer by chlorophyllb in detergent micelles

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyllb-containing detergent micelles with Triton X-100 were studied in a concentration range ofc = 0.4 μM–0.6mM chlorophyllb andc_d = 0.4–7.0mM Triton X-100. The concentration-dependent depolarization obeys Fo¨rster's theory of depolarization of fluorescence with a transfer distance parameterR₀ = 43 ± 2A?. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yieldη/η₀=sol1[1 + (cc_1/2)²] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149–161). With increasing chlorophyll b concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10 nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyllb molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyllb →chlorophyllb homotransfer may be about 3–26 % of the homotransfer chlorophylla →chlorophyll-a depending on the ratio of their concentrations.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

On photoabsorption of the neutral form of the green fluorescent protein chromophore

We present results of theoretical studies of the photoabsorption band corresponding to the vertical electronic transition S₀–S₁ between first two singlet states of the model chromophore from the green fluorescent protein (GFP) in its neutral form. Predictions of quantum chemical approaches including ab initio and semi-empirical approximations are compared for the model systems which mimic the GFP chromophore in different environments. We provide evidences that the protein matrix in GFP accounts for a fairly large shift of about 40 nm in the S₀–S₁ absorption band as compared to the gas phase.     

Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome c oxidase from bovine heart

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca²⁺ corresponds actually to a first derivative (differential) of the heme a ²⁺ absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a ²⁺ a ₃ ³⁺ -CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced heme a in the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca²⁺. The spectrum of heme a ²⁺ in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ～10 nm (589 cm^?1) corresponding to the perpendicularly oriented electronic π→π* transitions B _0x and B _0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of heme a ²⁺ originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838–848). The differences indicate that the overall γ-band of heme a ²⁺ in cytochrome oxidase contains in addition to the B _0x and B _0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of heme a ²⁺ contains significant contribution from heme a ₃ ²⁺ . The reconstructed absorption band of heme a ²⁺ in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm^?1) corresponds most likely to the individual Q _0y transition of heme a, whereas the Q _0x transition contributes only weakly to the spectrum.     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

Effects of removal and reconstitution of the extrinsic 33, 24 and 16 kDa proteins on flash oxygen yield in Photosystem II particles

The effects of removal and reconstitution of the three extrinsic proteins on the flash O₂ yield were investigated and the following results were obtained. (1) Removal in darkness of the 24 and 16 kDa proteins affected neither the oscillation pattern nor the signal amplitude of the flash O₂ yield. However, the signal amplitude was reduced with a factor of 2 in the presence of EDTA and was restored by excess Ca²⁺. The EDTA treatment did not change the oscillation pattern of the flash O₂ yield, but considerably damped the oscillation pattern of thermoluminescence B band. These results suggest a heterogeneity among the centers in binding affinity for Ca²⁺, and that Ca²⁺ removal induces an all-or-none type inactivation of O₂ evolution but not in the thermoluminescence processes, indicative of an inhibition of the S-state turnover at a specific S-state. (2) Removal in darkness of the 33, 24 and 16 kDa proteins abolished the flash O₂ yield, but the inhibited yield was appreciably restored either by reconstitution with the 33 kDa protein or by inclusion of 200 mM Cl⁻ in the reaction mixture. The flash O₂ yield reconstituted by the 33 kDa protein exhibited a rather normal oscillation pattern accompanied by a slightly increased damping, which could be simulated by assuming a high miss factor (30%) for S₃ → S₀ transition. The Cl⁻-restored flash O₂ yield exhibited a strongly damped oscillation pattern with obscured maxima at the 4th and 8th flashes, which was simulated by assuming a much higher miss factor (70%) for S₃ → S₀ transition. It was indicated that the Cl⁻-restored O₂ evolution considerably differs from the 33 kDa protein-reconstituted O₂ evolution with respect to the mechanism of S-state turnover.     

Absorbance difference spectra of the successive redox states of the oxygen-evolving apparatus of photosynthesis

The spectra of the absorbance changes due to the turnover of the so-called S-states of the oxygen-evolving apparatus were determined. The changes were induced by a series of saturating flashes in dark-adapted Photosystem II preparations, isolated from spinach chloroplasts. The electron acceptor was 2,5-dichloro-p-benzoquinone. The fraction of System II centers involved in each S-state transition on each flash was calculated from the oscillation pattern of the 1 ms absorbance transient which accompanies oxygen release. The difference spectrum associated with each S-state transition was then calculated from the observed flash-induced difference spectra. The spectra were found to contain a contribution by electron transfer at the acceptor side, which oscillated during the flash series approximately with a periodicity of two and was apparently modulated to some extent by the redox state of the donor side. At the donor side, the S₀ → S₁, S₁ → S₂ and S₂ → S₃ transitions were all three accompanied by the same absorbance difference spectrum, attributed previously to an oxidation of Mn(III) to Mn(IV) (Dekker, J.P., Van Gorkom, H.J., Brok, M. and Ouwehand, L. (1984) Biochim. Biophys. Acta 764, 301–309). It is concluded that each of these S-state transitions involves the oxidation of an Mn(III) to Mn(IV). The spectrum and amplitude of the millisecond transient were in agreement with its assignment to the reduction of the oxidized secondary donor Z⁺ and the three Mn(IV) ions.     

Femtosecond pump-supercontinuum probe and transient lens spectroscopy of adonixanthin

The ultrafast internal conversion (IC) dynamics of adonixanthin in organic solvents were studied by pump-supercontinuum probe (PSCP) and transient lens (TL) spectroscopy after photoexcitation to the S₂ state. Transient PSCP spectra in the range 344-768 nm provided the spectral evolution of the S₀ → S₂ ground state bleach and S₁ → S_n excited state absorption. Time constants were τ₂ = 115 and 111 fs for the S₂ → S₁ IC and τ₁ = 6.4 and 5.8 ps for the S₁ → S₀ IC in acetone and methanol, respectively. There was only an insignificant polarity dependence of τ₁, underlining the negligible importance of intramolecular charge transfer (ICT) in the lowest-lying excited state of C₄₀ carotenoids with carbonyl substitution on the β-ionone ring. A blueshift and a spectral narrowing of the S₁ → S_n ESA band, likely due to solvation dynamics, and formation of the adonixanthin radial cation at high pump energies via resonant two-photon ionization were found.