Light-Induced Damage of Histidine Residues in Photosystem II Reaction Center D1/D2/cyt b559 Complex

Histidine residue content of photosystem Ⅱ reaction center D1/D2/cytochrome b559 complex decreased by about 26% after illumination. The result suggests that some histidine residues are damaged by illumination. The damage of histidine residues may be related to the changes of the spectra properties during the incubation in the dark following preillumination of the reaction center complex.     

Reconstitution of plastoquinone in the D1/D2/cytochrome b-559 photosystem II reaction centre complex

Reconstitution of plastoquinone in the photosystem II D1/D2/cytochrome b-559 reaction centre complex, in the presence of the detergent Triton X-100, is reported. Illumination of the reconstituted system results in the reduction of cytochrome b-559, the process being partly herbicide-sensitive. In addition, the reconstitution of plastoquinone results in the ability of the isolated reaction centre to catalyse the photoreduction of 2,6-dichlorophenolindophenol in the presence of the exogenous electron donor diphenylcarbazide.     

Spectroelectrochemistry of cytochrome b559 in the D1-D2-Cyt b559 complex from spinach

The redox potential of cytochrome b559 (Cyt b559) in the D1-D2-Cyt b559 complex from spinach has been determined to be +90+/-2mV vs. SHE at pH 6.0, by thin-layer cell spectroelectrochemistry for the first time. The redox potential, corresponding uniquely to the so-called "low-potential form", exhibited a sigmoidal pH-dependence from pH 4.0 to 9.0, ranging from +115 to +50mV. An analysis of the pH-dependence based on model equations suggests that two histidine residues coordinating to the heme iron in the protein subunits may exert electrostatic influence on the redox potential of Cyt b559.     

Fluorescence Decaying Kinetics and Photodamage of Quinone-Reconstituted D1/D2/Cytochrome b559 Complex

Photosystem Ⅱ reaction center D1/D2/Cytochrome b559 complex loses its bound secondary electron acceptor QA and QB during isolation and purification. The artificial plastoquinone can reconstitute with the complex. The reconstitution of decyl-plastoquinone (DPQ) with D1/D2/Cytochrome b559 complex results in a decrease of the fraction of the two long lived fluorescence decay components (24 ns and 73 ns) coupled with photochemical activities to the total fluorescence yields, as well as a decrease of the total fluorescence intensity and a blue-shift of maximum emission wavelength. These results suggest that as the electron acceptor of reduced Pheo, DPQ restricts the charge recombination of P680+ Pheo-, and the two long lived fluorescence decay components (24 ns and 73 ns) come from the recombination. Although DPQ reconstitution has little effect on the susceptibility of Chi a to photodamage, β-carotene can easily be photodamaged after DPQ reconstitution. This is probably related to the physiological function of β-carotene.     

The identification of the Photosystem II reaction center: a personal story

This minireview is about the path that led me to the identification of the Photosystem II reaction center in oxygenic photosynthesis. It is based mostly on my own experiences and viewpoints. Thus, the article is essentially a personal account, and does not include all contributions that led to the identification of this functional unit of Photosystem II. This revised version was published online in August 2006 with corrections to the Cover Date.     

质体醌重组的D1／D2／Cytb559复合物的荧光衰减动力学和光破坏作用

光系统Ⅱ反应中心Ｄ１／Ｄ２／Ｃｙｔｂ５５９ 在分离纯化过程中失去了电子受体ＱＡ 和ＱＢ,人工合成的质体醌可以与Ｄ１／Ｄ２／Ｃｙｔｂ５５９ 复合物发生重组。癸基质体醌（ＤＰＱ）与Ｄ１／Ｄ２／Ｃｙｔｂ５９９ 复合物的重组导致该复合物的荧光强度下降及发射光谱蓝移,同时两个与光化学活性相关的长寿命（２４ ｎｓ和７３ ｎｓ）荧光衰减组分占整个荧光的百分数下降,这些结果表明ＤＰＱ作为Ｐｈｅｏ－ 的电子受体,限制了Ｐ６８０＋ ·Ｐｈｅｏ－ 的电荷重组。ＤＰＱ 的加入对Ｄ１／Ｄ２／Ｃｙｔｂ５５９复合物中Ｃｈｌａ 分子的光破坏敏感性影响不大,但β－胡萝卜素在加入ＤＰＱ 之后可以被光照破坏,这个过程可能与β－胡萝卜素的生理功能相关。     

光系统Ⅱ反应中心复合物中Cytb559的光还原

以分离纯化的光系统Ⅱ反应中心D1/D2/Cyt b559复合物为实验体系,在厌氧条件下,观察到Cytb559的光还原,表明Cyt b559能直接从Pheo~-接受电子,而且Cyt b559的光还原是不可逆的。当外加次级电子受体2,6-二甲基苯醌(DMBQ)与D1/D2/Cyt b559复合物重组之后,Cyt b559的光还原被延迟了,此时电子主要通过DMBQ传递,而且还原的Cyt b559在光照后的暗放置中有部分氧化。作者认为不依赖于醌受体的由Pheo~-到Cyt b559的电子传递是一条新的、次要的电子传递路线,它对光系统Ⅱ反应中心起保护作用。     

Photoreduction of Cytochrome b559 in the Photosystem Ⅱ Reaction Center Complex

The isolated and purified photosystem Ⅱ (PS Ⅱ ) reaction center D1/D2/Cyt b559 complex was taken as the experimental system. It was observed that under anaerobic conditions, cytochrome b559 (Cyt b559) could be reduced by exposure to strong illumination, suggesting Cyt b559 could accept electrons directly from reduced pheophytin (Pheo-). And the photoreduction of Cyt b559 was irreversible. When the isolated D1/D2/Cyt b559 complex reconstituted with exogenous secondary electron acceptor 2,6-dimethyl-benzoquinone (DMBQ), the photoreduction of Cyt b559 was delayed in the function of illumination time. Meanwhile, the electrons transferred mainly through DMBQ and photoreduced Cyt b559 could be partially reoxidized in the dark incubation following illumination. It was concluded that the quinone-independent electron transfer via Cyt b559 was a new, secondary electron pathway, which represented one of the protective pathes for PS Ⅱ reaction center to dissipate excess excitation energy.     

Effect of Photosystem II inhibitor K-15 on photochemical reactions of the isolated D1/D2 cytochrome b559 complex

Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo^–) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 M) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 M and 2 M resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the variable part of chlorophyll fluorescence (which is the recombination luminescence of the pair P₆₈₀ ⁺ Pheo^–). A 50% inhibition was induced by 5 M K-15 and the effect was maximal in the range 20 to 200 M. Photooxidation of P₆₈₀ in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 M). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo^– and P₆₈₀ ⁺ (or Z⁺) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for Q_A missing in the preparation.At a concentration of 0.5–50 M K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl₂. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo^– to cytochrome b559.Abbreviations K-15 4-[methoxy-bis-(trifluoromethyl)methyl]-2,6-dinitrophenyl hydrazone methyl ketone - P₆₈₀ the primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A and Q_B the primary and the secondary electron acceptor of PS II - RC reaction centre - SiMo silicomolybdate - TMPD N,N,N,,N,-tetramethyl-p-phenylenediamine - Z secondary electron donor of PS II     

Heat-induced changes in the EPR signal of tyrosine D ($$ Y^{{OX}}_{D} $$): a possible role of Cytochrome b559

The present study for the first time describes a close relationship between a change in the states of Cyt b559, a damage to Mn complex and a rapid reduction of tyrosine D (Y_D) as a function of temperature in spinach thylakoid membranes. Measurements of the EPR signal of dark stable tyrosine D in heat-treated thylakoid membranes showed a gradual decay of the oxidized state of tyrosine D with the progression of temperature. Simultaneously, it leads to the conversion of high-potential Cytochrome b559 into its low-potential form. We have speculated a possible involvement of Cytochrome b559 in the primary reduction events of tyrosine D in dark at high temperature. However, rapid reduction of tyrosine D may also be due to the disassembly of the Mn clock, which causes exposure of Y_D to the lumen and thereby its reduction by some unknown factor. These conclusions are supported by the measurements of Mn²⁺ release and thermoluminescence curves of various charge pairs in heat-treated thylakoid membranes. The results reveal an important aspect on the role of Cyt b559 in PS II during temperature stress.     

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.     

Glycinebetaine protects the D1/D2/Cytb559 complex of photosystem II against photo-induced and heat-induced inactivation

The presence of 1.0 mol/L glycinebetaine during isolation of D1/D2/Cytb559 reaction centre (RC) complexes from photosystem II (PSII) membrane fragments preserved the photochemical activity, monitored as the light-induced reduction of pheophytin and electron transport from diphenylcarbazide to 2.6-dichlorophenol-indophenol.-Glycinebetaine also protected the D1/D2/Cytb559 complexes against strong light-induced damage to the photochemical reactions and the irreversible bleaching of beta-carotene and chlorophyll. The presence of glycinebetaine also enhanced thermotolerance of the D1/D2/Cytb559 complexes isolated in the presence of 1.0 mol/L betaine with an increase in the temperature for 50% inactivation from 29 degrees C to 35 degrees C. The results indicate an increased supramolecular structural stability in the presence of glycinebetaine.     

The enigmatic cytochrome b-559 of oxygenic photosynthesis

The ubiquitous and obligatory association of cytochrome b -559 with the photosystem II reaction center of oxygenic photosynthesis is a conundrum since it seems not to have a function in the primary electron transport pathway of oxygen evolution. A model for the cytochrome structure that satisfies the cis -positive rule for membrane protein assembly consists of two short, non-identical hydrophobic membrane-spanning polypeptides (α and β), each containing a single histidine residue, as ligands for the bridging heme prosthetic group that is on the side of the membrane opposite to the water splitting apparatus. The ability of the heterodimer, but not the single α-subunit, to satisfy the cis -positive rule implies that the cytochrome inserts into the membrane as a heterodimer, with some evidence implicating it as the first membrane inserted unit of the assembling reaction center. The very positive redox potential of the cytochrome can be explained by a position for the heme in a hydrophobic niche near the stromal aqueous interface where it is also influenced by the large positive dipole potential of the parallel α-helices of the cytochrome. The requirement for the cytochrome in oxygenic photosynthesis may be a consequence of the presence of the strongly oxidizing reaction center needed for H₂O-splitting. This may lead to the need, under conditions of stress or plastid development, for an alternate source of electrons when the H₂O-splitting system is not operative as a source of reductant for the reaction center.     

The effect of hydration on protein flexibility in photosystem II of green plants studied by quasielastic neutron scattering

The effect of hydration on protein dynamics in photosystem II (PS II) membrane fragments from spinach has been investigated by using the method of quasielastic neutron scattering (QENS) at room temperature. The QENS data obtained indicate that the protein dynamics is strongly dependent on the extent of hydration. In particular, the hydration-induced activation of localized diffusive protein motions and Q_A⁻ reoxidation by Q_B in PS II appear to be correlated in their onset at a hydration value of about 45% relative humidity (r.h.). These findings underline the crucial functional relevance of localized diffusive protein motions on the picosecond-timescale for the reactions of light-induced photosynthetic water splitting under formation of plastoquinol and molecular oxygen in PS II of green plants. Advanced neutron scattering and complementary techniques to study biological systems. Contributions from the meetings, “Neutrons in Biology”, STFC Rutherford Appleton Laboratory, Didcot, UK, 11–13 July and “Proteins At Work 2007”, Perugia, Italy, 28–30 May 2007.     

Observation of One Pheophytin a Molecule Not Associated with Primary Photochemistry in the Isolated Photosystem Ⅱ Reaction Center D1/D2/Cyt b559 Complex

Photodamage of pheophytin a (pheo a) in the isolated photosystem Ⅱ (PSⅡ ) reaction center D1/D2/Cyt b559 complex from spinach has been investigated by high performance liquid chromatographic method in detail. The results showed that: (1) There is one pheo a molecule which is not associated with the primary photochemistry in the PS Ⅱ reaction center complex. It may be considered that there are two different electron transfer branches in the PS Ⅱ reaction center just as in the purple bacterium photosynthetic reaction center. (2) The damaged pheo a may be attributed to the one bonding to the D2 protein comparing the D2 subunit in the PS Ⅱ reaction center with M subunit in the purple bacterium photosynthetic reaction center. (3) A possible arrangement model of redox cofactors in the PS Ⅱ reaction center was proposed based on our experiment.     

Light-Induced Damage of Pheophytin a Molecule in the Isolated Photosystem Ⅱ Reaction Center D1/D2/Cyt b559 Complex

Photodamage of some pigments in the isolated photosystem Ⅱ (PS Ⅱ ) reaction center D1/D2/Cyt b559 complex from spinach has been investigated by means of high performance liquid chromatography. The light-induced damage of pheophytin a (pheo a) in the complex was observed for the first time. The content of pheo a decreased about 47 % by illumination, suggesting only one of the two pheo a molecules in the PS Ⅱ reaction center complex was damaged. No damage of β-carotene was found.     

Kinetics of reactions involving C-550 and cytochrome b559 in chloroplasts at low temperature. Evidence for two photoreactions

The kinetics of reduction of C-550 and of oxidation of cytochrome b₅₅₉ are studied with spinach chloroplasts, at ?170°, under light-limited conditions, at different light intensities. The rate of reduction of C-550 is proportional to the light intensity I; the rate of oxidation of b₅₅₉ is 2–3 times slower and not proportional to I. We propose that two light reactions occur at the reaction center of Photosystem-II (RC-II) at low temperature.     

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.     

Inhibition of photosystem II photochemistry by Cr is caused by the alteration of both D1 protein and oxygen evolving complex

The effect of chromium (Cr) on photosystem II (PSII) electron transport and the change of proteins content within PSII complex were investigated. When Lemna gibba was exposed to Cr during 96 h, growth inhibition was found to be associated with an alteration of the PSII electron transport at both PSII oxidizing and reducing sides. Investigation of fluorescence yields at transients K, J, I, and P suggested for Cr inhibitory effect to be located at the oxygen-evolving complex and Q_A reduction. Those Cr-inhibitory effects were related to the change of the turnover of PSII D1 protein and the alteration of 24 and 33 kDa proteins of the oxygen-evolving complex. The inhibition of the PSII electron transport and the formation of reactive oxygen species induced by Cr were highly correlated with the decrease in the content of D1 protein and the amount of 24 and 33 kDa proteins. Therefore, functional alteration of PSII activity by Cr was closely related with the structural change within PSII complex.