pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 M ferricyanide or after Cyt b-559 photooxidation in the presence of 2 M CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m^-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 M DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - CCCP carbonylcyanide-m-chlorophenylhydrazone - DCBQ 2,6-dichloro-p-benzoquinone - FeCN ferricyanide - MV methyl viologen - P₆₈₀ Photosystem II reaction center Chl-a dimer CIW-DPB publication No. 1118.     

On the question of the identity of cytochrome b-560 in thylakoid stromal membranes

Stromal membranes enriched in PS I contain a low potential cytochrome with a reduced -band peak close to 560 nm. The identity of this cytochrome component has been ascribed either to a low potential form of the Photosystem II cytochrome b-559 or to a different cytochrome with a reduced -band of 560 nm. The half-bandwidth of the 560 nm component in stromal membranes is identical to that of purified cytochrome b-559. Western blots show that the stromal membranes contain an amount of PS II cytochrome b-559 -subunit that is more than sufficient to account for the cytochrome b-560 detected spectrophotometrically in these membranes. These immunochemical data and the similarity of (i) the spectral peaks, and (ii) the redox properties of low potential PS II cytochrome b-559 and the b-560 component, suggest that the simplest inference is that the cytochrome b-560 protein in stromal membranes is identical to the PS II cytochrome b-559.Abbreviations: A absorbance - cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - E_mx midpoint potential at pH x - hbw half-bandwidth - LP low potential - MD menadiol - MES 2-(N-morpholino)ethanesulfonic acid - MHQ methylhydroquinone - PS I-PS II photosystems I, II - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Enhanced susceptibility of the oxidized and unprotonated forms of high potential cytochrome b-559 toward DCMU

The nature of interaction of cytochrome b-559 high potential (HP) with electron transport on the reducing side of photosystem II was investigated by measuring the susceptibility of cytochrome b-559HP to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under different conditions. Submicromolar DCMU concentrations decreased the rate of absorbance change corresponding to cytochrome b-559HP photoreduction while the amplitude was lowered at higher concentrations (up to 10 M). Appreciable extents of cytochrome b-559HP photoreduction were observed at DCMU concentrations which completely abolished the electron transport from water to methyl viologen under the same experimental conditions. However, the susceptibility of cytochrome b-559HP to DCMU increased with the degree of cytochrome b-559HP oxidation, induced either by ferricyanide or by illumination of low intensity (2 W/m²) of red light in the presence of 2 M carbonyl cyanide-m-chlorophenylhydrazone. Also, the DCMU inhibition was more severe when the pH increased from 6.5 to 8.5, indicating that the unprotonated form of cytochrome b-559HP is more susceptible to DCMU. These results demonstrate that cytochrome b-559HP can accept electrons prior to the Q_B site, probably via Q_A although both Q_A and Q_B can be involved to various extents in this reaction. We suggest that the redox state and the degree of protonation of cytochrome b-559HP alter its interaction with the reducing side of photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - CCCP carbonylcyanide m-chlorophenylhydrazone - FeCN ferricyanide - HP high potential - MV methylviologen CIW-DPB Publication No.1096.     

The fate of cytochrome b559during anaerobic photoinhibition and its recovery processes

The function of the cytochrome b₅₅₉, a Photosystem II (PS II) reaction center ubiquitous component is not yet known. Cytochrome b₅₅₉appears in a high (HP) or low (LP) potential form. The HP form is converted into the LP form during aerobic photoinhibition. It has been proposed before that this conversion, assumed to be reversible, ascribes protection against light stress of PS II by redirecting electron flow within PS II thus avoiding charge recombination of the primary radical pair and related oxidative damage. Here, we have used an experimental system allowing to assay the relation between the cytochrome b₅₅₉redox potential shift, its reversibility and protection against light induced PS II inactivation. Under anaerobic conditions fast reversible photoinactivation of PS II in isolated spinach thylakoids is observed accompanied by monomerisation of PS II. Monomers did not dissociate further into PS II sub-particles and did not migrate out of the grana partitions as observed in aerobic photoinactivation. The anaerobic photoinactivation is accompanied by an increase in the cytochrome b₅₅₉LP/HP ratio. However, despite recovery of PS II activity and partially of its dimeric form in darkness under aerobic conditions, no reversal of the cytochrome b₅₅₉redox potential shift accompanied these processes. Re-exposure of reactivated thylakoids having an increased PS II population in the LP form of the cytochrome b₅₅₉to strong illumination under aerobic conditions, did not result in a measurable protection of PS II as compared to control thylakoids. While it is possible that cytochrome b₅₅₉may play a protective role against light stress in PS II, the results presented here do not indicate that the increase in the ratio LP/HP form is involved in this process.     

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized (“O-type” effects) or when it is reduced (“R-type” effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the “O-type” mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an “R-type” mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q_C which interacts with Cyt b559 and is clearly not the Q_B site. Binding of compounds L to the Q_C site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E_m of HP Cyt b559 with increasing concentration of L (up to 10 K_ox(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q_C site when HP Cyt b559 is reduced (described by K_red(L)) induce a conversion of HP Cyt b559 to lower potential redox forms (“R-type” transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q_C site are discussed.     

Characterisation of a H2O2-oxidisable cytochrome b-559 in intact chloroplasts with a new type of LED Array Spectrophotometer

Cytochrome (cyt) b-559 absorbance changes in intact chloroplasts were deconvoluted using a previously described LED-Array-Spectrophotometer (Klughammer et al. (1990), Photosynth Res 25: 317–327). When intact chloroplasts were isolated in the presence of ascorbate, approx. 15% of the total cyt b-559 could be transiently oxidised by 200 M H₂O₂ in the dark. This fraction displays low-potential properties, as it can be also oxidised by menadione in the presence of 5 mM ascorbate. Heat pretreatment increased the size of this fraction by a factor of 3–4. Low concentrations of cyanide (in the M range) prolonged the oxidation time while high concentrations suppressed the oxidation (I₅₀=1.5 mM KCN). The former KCN-effect relates to inhibition of ascorbate dependent H₂O₂-reduction which is catalysed by ascorbate peroxidase, whereas the latter effect reflects competition between H₂O₂ and CN^– for the same binding site at the cytochrome heme. In the light, much lower concentrations of H₂O₂ were required to obtain oxidation, the amplitude depending on light intensity and on the concentration of the added H₂O₂, but never exceeding approx. 15% of the total cyt b-559. In the light, but not in the dark, H₂O₂ also induced the transient oxidation of a cyt f fraction similar in size to the H₂O₂-oxidisable cyt b-559 fraction. In this case, H₂O₂ serves as an acceptor of Photosystem I in conjunction with the ascorbate peroxidase detoxification system. Light can also induce oxidation of a 15% cyt b-559 fraction without H₂O₂-addition, if nitrite is present as electron acceptor and the chloroplasts are depleted of ascorbate. It is concluded that light-induced cyt b-559 oxidation in vivo is likely to be restricted to the H₂O₂-oxidisable cyt b-559 LP fraction and is normally counteracted by ascorbate.Abbreviations APX ascorbate peroxidase - chl chlorophyll - cyt cytochrome - HP high potential - LP low potential - MDA monodehydroascorbate - PQ plastoquinone - PS I and PS II Photosystems I and II     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate     

Redox and spectral properties of cytochrome b559 in different preparations of photosystem II

A detailed analysis of the properties of cytochrome b(559) (Cyt b(559)) in photosystem II (PS II) preparations with different degrees of structural complexity is presented. It reveals that (i) D1-D2-Cyt b(559) complexes either in solubilized form or incorporated into liposomes contain only one type of Cyt b(559) with E(m) values of 60 +/- 5 and 100 +/- 10 mV, respectively, at pH 6.8; (ii) in oxygen-evolving solubilized PS II core complexes Cyt b(559) exists predominantly (>85%) as an LP form with an E(m,7) of 125 +/- 10 mV and a minor fraction with an E(m,7) of -150 +/- 15 mV; (iii) in oxygen-evolving PS II membrane fragments three different redox forms are discernible with E(m) values of 390 +/- 15 mV (HP form), 230 +/- 20 mV (IP form), and 105 +/- 25 mV (LP form) and relative amplitudes of 58, 24, and 18%, respectively, at pH 7.3; (iv) the E(m) values are almost pH-independent between pH 6 and 9.5 in all sample types except D1-D2-Cyt b(559) complexes incorporated into liposomes with a slope of -29 mV/pH unit, when the pH increases from 6 to 9.5 (IP and LP form in PS II membrane fragments possibly within a restricted range from pH 6.5 to 8); (v) at pH >8 the HP Cyt b(559) progressively converts to the IP form with increasing pH; (vi) the reduced-minus-oxidized optical difference spectra of Cyt b(559) are very similar in the lambda range of 360-700 nm for all types except for the HP form which exhibits pronounced differences in the Soret band; and (vii) PS II membrane fragments and core complexes are inferred to contain about two Cyt b(559) hemes per PS II. Possible implications of conformational changes near the heme group and spin state transitions of the iron are discussed.     

Thermoluminescence properties of the isolated photosystem two reaction centre

Formation of thermoluminescence signals is characteristics of energy- and charge storage in Photosystem II. In isolated D1/D2/cytochrome b-559 Photosystem II reaction centre preparation four thermoluminescence components were found. These appear at -180 (Z band), between -80 and -50 (Z_v band), at -30 and at +35°C. The Z band arises from pigment molecules but not correlated with photosynthetic activity. The Z_v and -30°C bands arise from the recombination of charge pairs stabilized in the Photosystem II reaction centre complex. The +35°C band probably corresponds to the artefact glow peak resulting from a pigment-protein-detergent interaction in subchloroplast preparations (Rózsa Zs, Droppa M and Horváth G (1989) Biochim Biophys Acta 973, 350–353).Abbreviations Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - D1 psbA gene product - D2 psbD gene product - P680 primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - RC reaction centre of PS II - TL thermoluminescence     

Functional size of Photosystem II determined by radiation inactivation

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy -rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the Q_B site: H₂O (Mn)₄ Y_z P680 Pheophytin Q phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the and subunits of cytochrome b ₅₅₉and the 4.5 kDa psbI gene product.Abbreviations Cyt cytochrome - PS Photosystem - P680 primary electron donor of Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II - Y_z tyrosine donor to P680     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

High and low potential states of the chloroplast cytochromeb-559 and thermodynamic control of non-cyclic electron transport

Most of the chloroplastb-559 is high potential at neutral pH as defined by hydroquinone reducibility. FCCP^* (20 M) and antimycin A (50 M) convert high potentialb-559 to a low potential state which can be reduced by ascorbate but not hydroquinone. The low and high potential states of cytochromeb-559 are different forms of the same cytochrome.Three lines of evidence indicate that the cytb-559 oxidized by photosystem I is low potential: (1) theb-559 photooxidized by far-red light in the presence of FCCP (3 M) is low potentialb-559; (2) the amplitude of theb-559 oxidation by far-red light and the amount of low potentialb-559 present in the dark have the same general dependence on pH; (3) inhibitor studies show that plastoquinone mediates the oxidation of cytb-559 by PS I.The well-known stimulation ofb-559 oxidation by far-red light in the presence of FCCP is attributed to FCCP-facilitated photoconversion of high potentialb-559 to a low potential form.It is concluded that if cytb-559 is oxidized by system I light, then it is a low potential form (E _m7+80 mV) which is oxidized. It is not proven, however, that a significant amount of cytb-559 is oxidized by PS I under coupled or physiological conditions.Possible thermodynamic regulation of non-cyclic electron flow involving the distribution between high and low potential forms of cytb-559 is discussed.     

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II membranes

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b₅₅₉ (cyt b₅₅₉) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP^Fe2+) form of cyt b₅₅₉, whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP^Fe3+) form of cyt b₅₅₉. Light-induced conversion of cyt b₅₅₉ from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b₅₅₉ after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b₅₅₉ proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b₅₅₉ depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b₅₅₉ is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.     

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu²⁺ ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO₄ and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (A of 430–440 nm) and by the flash induced formation of P680⁺ (A at 820 nm). The relatively slow relaxation kinetics of the latter signal (t_1/2 1 ms) may suggest that in a substantial fraction of the RCs Q_A remains bound to the complex.Abbreviations Car -carotene - Chl a chlorophyll a - CP43, CP47 chlorophyll-proteins, with R_m 43 and 47 kDa - DBMIB dibromothymoquinone,2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone - DM -dodecyl-d-maltoside - HPLC high-performance liquid chromatography - OG n-octyl--d-glucopyranoside - IMAC immobilied metal affinity chromatography - Pheo a pheophytin a - PQ-9 plastoquinone-9 - P680 primary electron donor in PS II - PS II RC Photosystem II reaction centre - Q_A primary electron acceptor in PS II - SB-12 N-dodecyl-N,N-dimethyl-3-amino-1-propanesulphonate, (sulphobetain 12)     

Photoinhibition in Chilling Stressed Wheat and Maize

At chilling stress, the contents of photosynthetic pigments decreased significantly in maize, but in wheat the contents of chlorophyll (Chl) remained unchanged whereas the contents of total carotenoids (Car) increased. In both species the contents of + carotene and lutein + lutein-5,6-epoxide remained unaffected, but the de-epoxidation state involving the components of the xanthophyll cycle increased. Under chilling stress the photosynthetic electron transport also displayed a general failure in maize but in wheat only photosystem (PS) 2 coupled to the water oxidation complex was inhibited. Moreover, in stressed maize the quinone pool decreased, while the low and high potential forms of cytochrome b ₅₅₉ increased. In wheat only the contents of cytochrome b _559LP decreased. Peroxidation of acyl lipids in the chloroplast lamellae became more distinct in chilling stressed maize but could also be detected in wheat. Thus in chilling stressed maize prevails an impairment of the acceptor site of PS2 while in wheat photodamage is restricted to the electron donation pathway from water to P680 or to the oxygen evolving complex.     

ESR spectroscopy demonstrates that cytochrome b559 remains low potential in Ca2+-reactivated,salt-washed PSII particles

Cytochrome b₅₅₉ in various Photosystem II preparations was studled by using low temperature ESR spectroscopy. This technique was used because it is able to distinguish high from low potential forms of the cytochrome owing to the g-value differences between these species. Moreover, by using low temperature irradiation to oxidize cyt b₅₅₉ we have avoided the use of redox mediators. Previous work (Ghanotakis DF., Topper J.N. and Yocum, C.F. (1984) Biochim. Biophys. Acta 767, 524–531) demonstrated that reduction and extraction of manganese of the oxygen evolving complex, which might be expected to alter the redox properties of cyt b₅₅₉, occurs when certain PSII preparations are exposed to reductants. The ESR data presented here show that a mixture of high potential and lower potential cyt b₅₅₉ species is observed in the oxygen evolving Photosystem II complex. Treatment of PSII membranes with 0.8 M Tris converts the high potential form(s) to those of lower potential. Exposure of the membranes to 2M NaCl shifts a significant amount of high potential cyt b₅₅₉ to lower potential form(s); addition of CaCl₂ reconstituted oxygen evolution activity but did not restore cyt b₅₅₉ to its high potential form(s).Abbreviations Chl chlorophyll - cyt cytochrome - DCBQ 2,5-dichloro-benzoquinone - DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone - ESR electron spin resonance - OEC oxygen evolving complex - PS photosystem Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Isolation and characterisation of the Photosystem two reaction centre complex from a double mutant of Chlamydomonas reinhardtii

A rapid procedure has been developed for the isolation of the photosystem two reaction centre complex (PS II RC) from a double mutant of Chlamydomonas reinhardtii, F54-14, which lacks the Photosystem one complex and the chloroplast ATPase. Thylakoid membranes are solubilised with 1.5% (w/v) Triton X-100 and the PS II RC purified by anion-exchange chromatography using TSK DEAE-650(S) (Merck). The complex has a pigment stoichiometry of approximately six chlorophyll a: two pheophytin a: one cytochrome b-559: one to two -carotene. It photoaccumulates reduced pheophytin and oxidised P680 in the presence of sodium dithionite and silicomolybdate, respectively. Immunoblotting experiments have confirmed the presence of the D1 and D2 polypeptides in this complex. The -subunit of cytochrome b-559 was identified by N-terminal sequencing. Comparison of the complex with the PS II RC from pea using SDS-polyacrylamide gel electrophoresis showed that their polypeptide compositions were similar. However, the -subunit of cytochrome b-559 from C. reinhardtii has a lower apparent molecular weight than the pea counterpart whereas the -subunit is larger.Abbreviations DM n-dodecyl -d-maltoside - RC reaction centre - SiMo silicomolybdate, SiMo₁₂O₄₀ ^4– - TAP Tris-acetate-phosphate     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ? in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM⁻¹ cm⁻¹ and 27.0 ± 1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.