A New Pathway of Photoinactivation of Photosystem II. Irreversible Photoreduction of Pheophytin Causes Loss of Photochemical Activity of Isolated D1–D2–Cytochrome b559 Complex

A new pathway of photoinactivation of photosystem II (PS II) connected with irreversible photoaccumulation of reduced pheophytin (Ph) in isolated D1–D2–cytochrome b ₅₅₉ complexes of reaction center (RC) of PS II was discovered. The inhibitory effects of white light illumination on photochemical activity of D1–D2–cytochrome b ₅₅₉ complexes of RCs of photosystem II, isolated from pea chloroplasts, have been compared under anaerobic conditions in the absence and in the presence of sodium dithionite, electron transfer from which to the oxidized primary electron donor P680⁺ results in the photoaccumulation of anion-radical of the primary electron acceptor, PH. In both cases, prolonged illumination (1-5 min, 120 W/m²) led to a pronounced loss of the photochemical activity as it was monitored by measuring the amplitude of the reversible photoinduced absorbance changes at 682 nm related to the photoreduction of Ph. The extent of the photoinactivation depended on the illumination time and pH of the medium. At pH 8.0, the presence of dithionite during photoinactivation brought about a protective effect compared to that in a control sample. In contrast, lowering pH to 6.0 increased the sensitivity to photoinactivation in the dithionite containing samples. For 5 min irradiation, the photochemical activity in the absence and in the presence of dithionite decreased by 35 and 72%, respectively (this was accompanied by an irreversible bleaching of the pheophytin Q_x absorption band at 542 nm). Degradation of the D1 and D2 proteins was not observed under these conditions. A subsequent addition of an electron acceptor, potassium ferricyanide, to the illuminated samples restored neither the amplitude of the signal at 682 nm nor absorption at 542 nm. It is suggested that at pH < 7.0 the photoaccumulated PH is irreversibly converted into a secondary, most probably protonated form, that does not lead to destruction of the RCs but prevents the photoformation of the primary radical pair [P680⁺PH]. A possible application of this effect to photoinactivation of PS II in vivo is discussed.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light （800μmol photons/m^2 per s）-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center （PSII RC） under aerobic conditions （in the absence of electron donors or acceptors） was studied using high-pressure liquid chromatography （HPLC）, absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSII RC as determined by HPLC after light treatment were as follows： with Increasing illumination time chlorophyll （Chl） a and β-carotene （β-car） content decreased. However, decreases in pheophytin （Pheo） could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illuminatlon, the initial bleaching occurred maximally at 680 nm but that with Increasing Illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed Initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 mln light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination. After Illumination, the fluorescence emission Intensity changed and the fluorescence maximum blue shifted, showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm^-1 （υ101）, 1 159 cm^-1 （υ2）, 1 006 cm^-1 （υ3）, 966 cm^-1 （υ4） for 488.0 nm excitation and 1 525 cm^-1 （υ1）, 1 159 cm^-1 （υ2）, 1 007 cm^-1 （υ3）, 968 cm^-1 （υ4） for 514.5 nm excitation. It was confirmed that two spectroscopically different β-car molecules exist In the PSII RC. After light treatment for 20 mln, band positions and bandwidths were unchanged. This indicates that carotenoid configuration Is not the parameter that regulates photoprotectlon in the PSII RC.     

CD Spectra of D1/D2/Cyt b559 Complax in Photodamaged Photosystem Ⅱ Reaction Center

The CD spectrum of photosystem Ⅱ reaction center D1/D2/Cyt b559 complex showed a strong reverse band with positive peak at 680 nm and negative peak at 660 nm in the red absorption region (Qy band). After the D1/D2/Cyt b559 complex was illuminated by strong light, the CD signals of the complex decreased significantly in the red region in which the negative peak still existed but the positive one disappeared. The result suggested that the CD signal of photosystem Ⅱ reaction center D1/D2/Cyt b559 complex not only came from the primary donor, P680, but also from other pigments such as from accessory Chl a or Pheo a.     

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.     

Light-induced Damage of Photosystem Ⅱ Primary Electron Donor P680: a High Performance Liquid Chromatographic Analysis of Pigment Content in D1/D2/Cytochrome b559 Complex Under Photoinhibitory Conditions

By HPLC analytical method, the change of PS Ⅱ RC' s pigment content in the process of photodamage under strong illumination from spinach ( Spinacia oleracea Mill. ) was comparatively studied. The experimental results show that: (1) In authors' analytical conditions, (of which, [Chl] = 150 µg/mL, and the illumination strength was put at 2.3 ×10 6 mJ·m－2·s－1 ), 45 rain of illumination could cause almost the whole loss of A680 in the fourth derivative absorption spectra, while A670 decreased to about one half of its original intensity; the absorption maximum in red, concurrently, was shifted from 676 nm to 671 nm, representing the loss of more than 90% of the photochemical activities of the PS Il RC. (2) During the period of continuous illumination, the Chl concentration decreased in a 3-period style, which meant that the first [Chl] decreased to the 2/3 of its original amount from 20 min to 40 rain after illumination had started, then became stabilized up to about 60 min of illumination, there after a second decrease of [ Chl ] in another about 20 min until it reached about 30 % of the original level and remained unchanged from about 80 min on. The original pigment components of D1/D2/Cyt b559 was approximately as 6 Chl a:2 Pheo:2β-Car which are in support of authors' previous proposal about the minimum Chl/Pheo ratio of 4: 2 in PS Ⅱ RC’s pigment contents. (3) After about 40 min of illumination, a newly appeared elution peak was found between the Pheo andβ-Car peaks in HPLC profile, at the retention time of 7.2 min, a little later than that (6.9 rain) of Pheo molecules, the newly appeared elution peak was supposed to be a kind of accumulated and stable product of the PS II RC's photodamage process and very much possible the Pheo-like molecules.     

Histidine residue 252 of the Photosystem II D1 polypeptide is involved in a light-induced cross-linking of the polypeptide with the α subunit of cytochrome b-559: study of a site-directed mutant of Synechocystis PCC 6803

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors Q_A and Q_B and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1-cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1-cytochrome adduct is the product of a reaction between the oxidized residue His²⁵² of the D1 polypeptide and the N-terminal amino group of the cytochrome α subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.     

Structure–function of the cytochrome b6f lipoprotein complex: a scientific odyssey and personal perspective

Photosynthesis Research - Structure–function studies of the cytochrome b6f complex, the central hetero-oligomeric membrane protein complex in the electron transport chain of oxygenic...     

Comparison of the cytochrome bc1 complex with the anticipated structure of the cytochrome b6f complex: Le plus ça change le plus c'est la même chose

Structural alignment of the integral cytochrome b6-SU IV subunits with the solved structure of the mitochondrial bc1 complex shows a pronounced asymmetry. There is a much higher homology on the p-side of the membrane, suggesting a similarity in the mechanisms of intramembrane and interfacial electron and proton transfer on the p-side, but not necessarily on the n-side. Structural differences between the bc1 and b6f complexes appear to be larger the farther the domain or subunit is removed from the membrane core, with extreme differences between cytochromes c1 and f. A special role for the dimer may involve electron sharing between the two hemes b(p), which is indicated as a probable event by calculations of relative rate constants for intramonomer heme b(p) --> heme b(n), or intermonomer heme b(p) --> heme b(p) electron transfer. The long-standing observation of flash-induced oxidation of only approximately 0.5 of the chemical content of cyt f may be partly a consequence of the statistical population of ISP bound to cytfon the dimer. It is proposed that the p-side domain of cyt f is positioned with its long axis parallel to the membrane surface in order to: (i) allow its large and small domains to carry out the functions of cyt c1 and suVIII, respectively, of the bc1 complex, and (ii) provide maximum dielectric continuity with the membrane. (iii) This position would also allow the internal water chain ("proton wire") of cyt f to serve as the p-side exit port for an intramembrane H+ transfer chain that would deprotonate the semiquinol located in the myxothiazol/MOA-stilbene pocket near heme b(p). A hypothesis is presented for the identity of the amino acid residues in this chain.     

Isolation and Characterization of Photosystem Ⅱ Reaction Center D1-D2-Cytochrome b-559 Complex from the Chloroplasts of Spinach

Photosystem Ⅱ reaction center D1-D2-cytochrome b-559 pigment-protein complex has been isolated and purified from chloroplasts of spinach and its properties have been studied. The Isotared photosystem II reaction center contains close to six chlorophyll a per two pheophytin a molecules. Analysis of fluorescence decaying by phase modulation fluorometry suggests that the reaction center has three components of fluorescence decaying with lifetimes of 1.5 nS, 6.23 nS, 36.26 nS in terms of fractions to total fluorescence yield as 0.06, 0.67, 0.27 respectively. The ,6.25 nS fluorescence component corresponds to chlorophyll a which is energetically uncoupled from the process of charge separation. The proportion of 1.51 nS component is very low, and its source is unclear. The 36.25 nS fluorescence component is attributed to the recombination of the primary radical pair, and so represents the activity of charge separation.     

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.     

Effect of concentration of iodide on the bound species of I2/I−3 in the amylose-iodine complex

A liquid-liquid distribution method, with heptane as the organic solvent, involving evaluation of the concentration of free 1⁻ by magnetic circular dichroism, has been developed for determining the bound amounts of I₂/I⁻₃ in the amylose-iodine complex in unbuffered aqueous solutions. The effect of I₂ and I⁻ concentrations on the bound species of iodine in the complex was investigated by using this method. We found that the stoichiometric bound species of I₂/I⁻₃ is independent of the concentration of I₂ at a given I⁻ concentration. However, the bound species strongly depends on I⁻ concentration, and varies from I⁻₃ at 10 mM KI to I⁻₁₅ at 0M KI. Moreover, the number of d-glucosyl residues required for including one iodine atom is within the range of 2.7 to 3.0, regardless of I⁻ concentration. It was concluded that the bound species are governed by the distribution of the actual species I₂·I₂ (I₄), (I₄), I₂·I⁻₃ (I⁻₅), and I⁻₃·I⁻₃ (I²⁻₆), which are responsible for the blue color of the complex.     

Tyrosine triad at the interface between the Rieske iron–sulfur protein,cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus

A triad of tyrosine residues (Y152–154) in the cytochrome c₁ subunit (C1) of the Rhodobacter capsulatus cytochrome bc₁ complex (BC1) is ideally positioned to interact with cytochrome c₂ (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations—A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.     

The Resonance Raman Spectra of β-Carotene Molecules in the Photosystem Ⅱ Reaction Center D1/D2/Cyt b-559 Complex

The resonance Raman spectrum of β-carotene in photosystem Ⅱ (PS Ⅱ )reaction center complex was characterized by four main bands, peaking at 1532 (νl), 1156 (ν2), 1010 (ν3) and 970 (ν4) cm -1, respectively, with several additional small Raman bands in the region between 1100 cm-1 and 1500 cm-1 It was suggested that β-carotene molecules of the reaction center complex were in all-trans configuration. The resonance Raman spectrum of an acetone extract from the reaction center complex also showed four main bands. The peak position of νl, ν3 and ν4 band shifted 5 cm-1 to the shorter wave number. The most dramatic changes were the reduction of the intensity of ν4. From the above results it was demon- strated that the conformation of β-carotene molecules in the PS Ⅰ reaction center was not the same as that of free β-carotene molecules in solution, but similar to that of carotenoid molecules in the photosynthetic bacterial reaction center, in other words, they are likely to be in a twisted conformation.     

Solubilization of green plant thylakoid membranes with n-dodecyl-α,D-maltoside. Implications for the structural organization of the Photosystem II,Photosystem I,ATP synthase and cytochrome b6 f complexes

A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid membranes from spinach with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b ₆ f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms. Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes. An extensive analysis by electron microscopy and image analysis of the CF₀F₁ ATP synthase complex suggests locations of the δ (on top of the F₁ headpiece) and ∈ subunits (in the central stalk) and reveals that in a substantial part of the complexes the F₁ headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure and may represent the complex in its inactive, oxidized form. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quality control of Photosystem II under light stress – turnover of aggregates of the D1 protein in vivo

When photodamaged under excessive light, the D1 protein is digested and removed from Photosystem (PS) II to facilitate turnover of the protein. In vitro studies have shown that part of the photodamaged D1 protein forms aggregates with surrounding polypeptides before being digested by a protease(s) in the stroma [Yamamoto Y (2001) Plant Cell Physiol 42: 121–128]. The aim of this study was to examine whether light-induced aggregation of the D1 protein also occurs in vivo. The following results were obtained: (1) PS II activity in spinach leaves was significantly inhibited by weak illumination (light intensity, 20–100 μE m⁻² s⁻¹), as monitored by chlorophyll fluorescence Fv/Fm, when the leaves were kept at higher temperatures (35–40 °C); (2) aggregation of the D1 protein, as well as cleavage of the protein, was detected in thylakoids isolated from spinach leaves that had been subjected to heat/light stress; (3) aggregates of the D1 protein disappeared after incubation of the leaves at 25 °C in the dark or under illumination with weak light. Since it is dependent on the presence of oxygen, aggregation of the D1 protein is probably induced by reactive oxygen species produced in thylakoids upon illumination at elevated temperatures. Consistent with this notion, singlet oxygen production in thylakoid samples under illumination was shown to be stimulated significantly at higher temperatures.     

Sensitivity of the photosynthetic apparatus to UV-A radiation: role of light-harvesting complex II–photosystem II supercomplex organization

In this work we study the effect of UV-A radiation on the function of the photosynthetic apparatus in thylakoid membranes with different organization of the light-harvesting complex II–photosystem II (LHCII–PSII) supercomplex. Leaves and isolated thylakoid membranes from a number of previously characterized pea species with different LHCII size and organization were subjected to UV-A treatment. A relationship was found between the molecular organization of the LHCII (ratio of the oligomeric to monomeric forms of LHCII) and UV-A-induced changes both in the energy transfer from PSII to PSI and between the chlorophyll–protein complexes within the LHCII–PSII supercomplex. Dependence on the organization of the LHCII was also found with regard to the degree of inhibition of the photosynthetic oxygen evolution. The susceptibility of energy transfer and oxygen evolution to UV-A radiation decreased with increasing LHCII oligomerization when the UV-A treatment was performed on isolated thylakoid membranes, in contrast to the effect observed in thylakoid membranes isolated from pre-irradiated pea leaves. The data suggest that UV-A radiation leads mainly to damage of the PSIIα centers. Comparison of membranes with different organization of their LHCII–PSII supercomplex shows that the oligomeric forms of LHCII play a key role for sensitivity to UV-A radiation of the photosynthetic apparatus. S. G. Taneva is Associated member of the Institute of Biophysics, Bulgarian Academy of Sciences.     

Mössbauer spectra of the heme peptide (HP) 1–50 and the heme peptide:non-heme peptide (NHP) non-covalent complex 1–50:51–104 derived from cytochrome c: evidence for cytochrome c iron site solvation in aqueous solution

Mössbauer spectroscopic studies on a heme peptide (HP) derived from cytochrome c and on the HP recombined non-covalently with the remaining cleaved section are reported. The results suggest that the environment of the heme site in the known crystal structure of cytochrome c may differ in detail from the environment of the heme in the working protein.