Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.     

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

Redox potentials of chlorophylls in the photosystem II reaction center

Water oxidation generating atmospheric oxygen occurs in photosystem II (PSII), a large protein-pigment complex located in the thylakoid membrane. The recent crystal structures at 3.2 and 3.5 A resolutions provide novel details on amino acid side chains, especially in the D1/D2 subunits. We calculated the redox potentials for one-electron oxidation of the chlorophyll a (Chla) molecules in PSII, considering the protein environment in atomic detail. The calculated redox potentials for the dimer Chla (P(D1/D2)) and accessory Chla (Chl(D1/D2)) were 1.11-1.30 V relative to the normal hydrogen electrode at pH 7, which is high enough for water oxidation. The D1/D2 proteins and their cofactors contribute approximately 390 mV to the enormous upshift of 470 mV compared to the redox potential of monomeric Chla in dimethylformamide. The other subunits are responsible for the remaining 80 mV. The high redox potentials of the two accessory Chla Chl(D1/D2) suggests that they also participate in the charge separation process.     

Measurement of stoichiometry of photosystem II to photosystem I reaction centers

A wide range of values for the photosystem II to photosystem I stoichiometry have been reported. It is likely that some of this variation is due to measurement artifacts, which are discussed. Careful measurements of photosystem II reactions by absorption change at 325 nm, and flash yields of oxygen evolution, of protons from oxidation of water and of reduction of dichloroindophenol give equivalent results. Stoichiometries other than 1:1 are routinely found, and they vary with growth conditions as well as plant type. Two atrazine binding sites are found for every photosystem II reaction center that is active in oxygen evolution.     

Comparison of bacterial reaction centers and photosystem II

In photosynthetic organisms, the utilization of solar energy to drive electron and proton transfer reactions across membranes is performed by pigment-protein complexes including bacterial reaction centers (BRCs) and photosystem II. The well-characterized BRC has served as a structural and functional model for the evolutionarily-related photosystem II for many years. Even though these complexes transfer electrons and protons across cell membranes in analogous manners, they utilize different secondary electron donors. Photosystem II has the unique ability to abstract electrons from water, while BRCs use molecules with much lower potentials as electron donors. This article compares the two complexes and reviews the factors that give rise to the functional differences. Also discussed are the modifications that have been performed on BRCs so that they perform reactions, such as amino acid and metal oxidation, which occur in photosystem II.     

Excitonic interactions in the reaction centre of photosystem II studied by using circular dichroism

Changes in excitonic interactions of photosystem II (PSII) reaction centre (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor (pheophytin (Pheo)) were analysed using circular dichroism (CD). The CD spectrum of PSII RC shows positive bands at 417, 435 and 681 and negative bands at 447 and 664 nm. Oxidation of the primary donor by illuminating the sample in the presence of silicomolybdate resulted in nearly symmetric decrease of CD amplitudes at 664 and 684 nm. In the Soret region, the maximum bleaching of CD signal was detected at 449 and 440 nm. Accumulation of reduced Pheo in the presence of dithionite brought about much lower changes in CD amplitudes than P680 oxidation. In this case, only a small asymmetric bleaching at 680 and 668 nm in the red region and a bleaching at 445, 435 and 416 nm in the Soret region has been detected. Therefore, we suppose that the contribution of the Pheo of the primary acceptor to the total CD signal of RC is negligible. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was strongly temperature-dependent. The reversible CD bleaching was completely inhibited below 200 K, although the reduced Pheo was accumulated even at a temperature of 77 K. Since the temperature does not influence the excitonic interaction, the temperature dependence of the CD changes upon Pheo reduction does not support the model of Pheo excitonically interacting with the other chlorophylls (Chl) of the RC. We propose that Pheo should not be considered as a part of a multimer model.     

Excitonic interactions in the reaction centre of photosystem II studied by using circular dichroism

Changes in excitonic interactions of photosystem II (PSII) reaction centre (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor (pheophytin (Pheo)) were analysed using circular dichroism (CD). The CD spectrum of PSII RC shows positive bands at 417, 435 and 681 and negative bands at 447 and 664 nm. Oxidation of the primary donor by illuminating the sample in the presence of silicomolybdate resulted in nearly symmetric decrease of CD amplitudes at 664 and 684 nm. In the Soret region, the maximum bleaching of CD signal was detected at 449 and 440 nm. Accumulation of reduced Pheo in the presence of dithionite brought about much lower changes in CD amplitudes than P680 oxidation. In this case, only a small asymmetric bleaching at 680 and 668 nm in the red region and a bleaching at 445, 435 and 416 nm in the Soret region has been detected. Therefore, we suppose that the contribution of the Pheo of the primary acceptor to the total CD signal of RC is negligible. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was strongly temperature-dependent. The reversible CD bleaching was completely inhibited below 200 K, although the reduced Pheo was accumulated even at a temperature of 77 K. Since the temperature does not influence the excitonic interaction, the temperature dependence of the CD changes upon Pheo reduction does not support the model of Pheo excitonically interacting with the other chlorophylls (Chl) of the RC. We propose that Pheo should not be considered as a part of a multimer model.     

A light-induced spin-polarized triplet detected by EPR in photosystem II reaction centers

A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.     

Mechanism of photoinduced electron transfer in photosystem II reaction centers

The shape of the EPR spectrum of the triplet state of photosystem II reaction centers with a singly reduced primary acceptor complex Q_AFe²⁺ was studied. It was shown that the spectroscopic properties do not significantly change when the relaxation of the primary acceptor is accelerated and when the magnetic interaction between the reduced quinone molecule Q_A and the nonheme iron ion Fe²⁺ is disrupted. This observation confirmed the earlier conclusion that the anisotropy of the quantum yield of the triplet state is the main cause of the anomalous shape of the EPR spectrum. A scheme of primary processes in photosystem II that is consistent with the observed properties of the EPR spectrum of the triplet state is discussed.     

On the mechanism of photoinduced electron transfer in photosystem II reaction centers

The EPR spectrum of the triplet state of photosystem II reaction centers has been studied in the case of the singly reduced primary acceptor complex QAFe2+. It was demonstrated that the shape of the spectrum does not change much when the relaxation of the primary acceptor is accelerated and when magnetic interaction between the reduced quinone molecule QA and the non-heme iron Fe2+ is disrupted. This observation confirms the earlier conclusion that the anomalous shape of the EPR spectrum is due mainly to the anisotropy of the quatum yield of the triplet state. A scheme of primary events in photosystem II is discussed, which is consistent with the observed properties of the EPR spectrum of the triplet state.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Reversible inactivation of photosystem II reaction centers in cation-depleted chloroplast membranes

Isolated pea chloroplasts were washed once in 10 mm NaCl and were then suspended in “low-salt” medium. Approximately one-half of the photosystem II reaction centers of these salt-depleted membranes were found to be photochemically inactive. These units became active in the presence of low concentrations of divalent cations (5–10 mm Mg²⁺) or high concentrations of monovalent cations (150–200 mm Na⁺), as evidenced by a twofold increase in the steady-state flash yield of oxygen evolution under short (~10-μs) saturating repetitive flashes (two per second). The half-maximal increase in flash yield occurred at ~2 mM Mg²⁺ or ~75 mm Na⁺. The flash yield of hydroxylamine oxidation in these low-salt chloroplasts increased twofold after Mg²⁺ addition, indicating that the cation action was close to the reaction-center chlorophyll complex. The relation between flash yield and dark time between flashes was not changed significantly by Mg²⁺, indicating that the rate-limiting step of the overall electron transport (H₂0 —→ ferricyanide) was not affected significantly. When the rate-limiting step was bypassed using silicomolybdate as the photosystem II electron acceptor (in the presence of diuron), the reduction rate doubled in the presence of Mg²⁺, even under continuous, saturating light. In glutaraldehyde-fixed chloroplasts, Mg²⁺ did not increase the flash yield of O² evolution; this suggests that protein conformational changes in the chloroplast membranes were involved in Mg²⁺ activation of photosystem II centers.     

Light saturation response of inactive photosystem II reaction centers in spinach

Oxygen evolving photosystem II particles were exposed to 100 and 250 W m^–2 white light at 20°C under aerobic, anaerobic and strongly reducing (presence of dithionite) conditions. Three types of photoinactivation processes with different kinetics could be distinguished: (1) The fast process which occurs under strongly reducing (t _1/21–3 min) and anaerobic conditions (t _1/24–12 min). (2) The slow process (t _1/215–40 min) and (3) the very slow process (t _1/2>100 min), both of which occur under all three sets of conditions.The fast process results in a parallel decline of variable fluorescence (F _v) and of Hill reaction rate, accompanied by an antiparallel increase of constant fluorescence (F _o). We assume that trapping of Q_A in a negatively charged stable state, (Q_A ^–)_stab, is responsible for the effects observed.The slow process is characterized by a decline of maximal fluorescence (F _m). In presence of oxygen this decline is due to the well known disappearance of F _v which proceeds in parallel with the inhibition of the Hill reaction; F _o remains essentially constant. Under anaerobic and reducing conditions the decline of F _m represents the disappearance of the increment in F _o generated by the fast process. We assume that the slow process consists in neutralization of the negative charge in the domain of Q_A in a manner that renders Q_A non-functional. The charge separation in the RC is still possible, but energy of excitation becomes thermally dissipated.The very slow photoinactivation process is linked to loss of charge separation ability of the PS II RC and will be analyzed in a forthcoming paper.Abbreviations F chlorophyll a fluorescence - F _o, F _v, F _m constant, variable, maximum fluorescence - F _o, F _v, F _m the same, measured in presence of dithionite (F _v suppression method) - PS II photosystem II - RC reaction centre (P680. Pheo) - P680 primary electron donor - Pheo pheophytin, intermediary electron acceptor - Q_A, Q_B the primary and secondary electron acceptor - Z, D electron donors to P680 - (Q_A)_stab, (Q_A H)_stab hypothetical modifications of Q_A resulting from photoinactivation - O-, A- and R-conditions aerobic, anaerobic and strongly reducing (presence of dithionite) conditions - MES 2-(N-morpholine) ethanesulphonic acid - DCPIP 2,6-dichlorphenolindophenol - GGOC mixture of glucose, glucose oxidase and catalase - DT-20 oxygen-evolving PS II particles     

Pheophytin-protein interactions in photosystem II studied by resonance Raman spectroscopy of modified reaction centers

Soret-excited resonance Raman spectra of two types of pheophytin-exchanged photosystem II RCs are reported. The cofactor composition of the reaction centers was modified by exchanging pheophytin a for 13(1)-deoxo-13(1)-hydroxypheophytin a, yielding one preparation with selective replacement of the photochemically inactive pheophytin (H(B)) and a second one exhibiting total replacement of H(B) and 40% replacement of H(A), the primary electron acceptor. Resonance Raman spectra indicate that the other bound cofactors present are not significantly perturbed by Pheo substitution. The resonance Raman contributions from H(A) and H(B) in the carbonyl stretching region are identified at 1679 and 1675 cm(-)(1), respectively, indicating that both pheophytin molecules in the photosystem II reaction center have hydrogen-bonded keto-carbonyl groups. This conclusion differs from what is observed in the functionally related RCs of purple non-sulfur bacteria, where the keto-carbonyl group of H(B) is not hydrogen bonded, but confirms predictions from models based on protein sequence alignments.     

Heterogeneity of photosystem II reaction centers as influenced by heat treatment of barley leaves

Three functionally distinct populations of PSII reaction centers differing in the ability to keep the primary acceptors in a reduced state and to transfer electrons to PSI were estimated using chlorophyll fluorescence measurements in primary barley leaves exposed to elevated temperatures in the range of 37–51°C. The capacity of the PSII reaction centers to perform at least one light-induced charge separation was not affected by a 5-min heat treatment at temperatures up to 51°C. The first population containing Q_B-non-reducing centers corresponded to 15–20% of the total PSII activity up to 45°C. In a second population, PSII reaction centers maintained Q_A reduction under light in the presence of oxygen, but not in the presence of a strong artificial PSI electron acceptor, methyl viologen. In a third population that gradually increases from zero at 37°C to about 60% at 45°C, the PSII centers were not able to keep Q_A in the reduced state even in the presence of oxygen as the sole electron acceptor. Three electron transport pathways, the pseudocyclic one involving both PSII and PSI, the NAD(P)H-dependent pathway mediated by PSI alone after the loss of activity in some PSII centers, and the PSI-driven ferredoxin-dependent route enhanced by weakly efficient PSII centers that are able to provide only catalytic amounts of electrons, are suggested to create a proton gradient in chloroplasts of heat-stressed leaves thus protecting PSII reaction centers from photodamage.     

Electric field effects on the chlorophylls,pheophytins, and beta-carotenes in the reaction center of photosystem II

We present an electric field modulated absorption spectroscopy (Stark effect) study of isolated photosystem II reaction center complexes, including a preparation in which the inactive pheophytin H(B) was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin. The results reveal that the Stark spectrum of the Q(x) and Q(y) transitions of the pheophytins has a second-derivative line shape, indicating that the Stark effect is dominated by differences in the dipole moment between the ground and the electronically excited states of these transitions (Delta mu). The Delta mu values for the Q(x) and Q(y) transitions of H(B) are small (Delta mu = 0.6-1.0 D f(-1)), whereas that of the Q(x) transition of the active pheophytin H(A) is remarkably large (Delta mu = 3 D f(-1)). The Stark spectrum of the red-most absorbing pigments also shows a second-derivative line shape, but this spectrum is considerably red-shifted as compared to the second derivative of the absorption spectrum. This situation is unusual but has been observed before in heterodimer special pair mutants of purple bacterial reaction centers [Moore, L. J., Zhou, H., and Boxer, S. G. (1999) Biochemistry 38, 11949-11960]. The red-shifted Stark spectra can be explained by a mixing of exciton states with a charge-transfer state of about equal energy. We conclude that the charge transfer state involves H(A) and its immediate chlorophyll neighbor (B(A)), and we suggest that this (B(A)(delta+)H(A)(delta-)) charge transfer state plays a crucial role in the primary charge separation reaction in photosystem II. In contrast to most other carotenes, the two beta-carotene molecules of the photosystem II reaction center display a very small Delta mu, which can most easily be explained by excitonic coupling of both molecules. These results favor a model that locates both beta-carotene molecules at the same side of the complex.     

Water oxidation in photosystem II

Photosynthesis Research - Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for...     

Inactive photosystem II centers: A resolution of discrepencies in photosystem II quantitation

The abundance of photosystem II in chloroplast thylakoid membranes has been a contentious issue because different techniques give quite different estimates of photosystem II titer. This discrepancy led in turn to disagreements regarding the stoichiometry of photosystem II to photosystem I in these membranes. We believe that the discrepancy in photosystem II quantitation is resolved by evidence which shows that a large population of photosystem II centers with negligible turnover rates are present in isolated thylakoid membranes as well as in normally developed leaves of healthy plants.     

Multiple redox-active chlorophylls in the secondary electron-transfer pathways of oxygen-evolving photosystem II

Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P680(+). We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and beta-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 and 814 nm and three slow-decaying bands at 810, 825, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl(+)). The fast-decaying bands observed after illumination at 20 K could be generated again by reilluminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b 559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl(+) and Car(+) species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P680(+) and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulfoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P680(+).