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     

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 s and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219I A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of Q_A ^– to Q_A, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m^–2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V G256A > L275F V219I > F255Y CW-15 WT. At 300 and 60 W m^–2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4–0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

---

     

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     

Primary charge separation in Photosystem II

In this Minireview, we discuss a number of issues on the primary photosynthetic reactions of the green plant Photosystem II. We discuss the origin of the 683 and 679 nm absorption bands of the PS II RC complex and suggest that these forms may reflect the single-site spectrum with dominant contributions from the zero-phonon line and a pronounced ∼80 cm⁻¹ phonon side band, respectively. The couplings between the six central RC chlorins are probably very similar and, therefore, a `multimer' model arises in which there is no `special pair' and in which for each realization of the disorder the excitation may be dynamically localized on basically any combination of neighbouring chlorins. The key features of our model for the primary reactions in PS II include ultrafast (<500 fs) energy transfer processes within the multimer, `slow' (∼20 ps) energy transfer processes from peripheral RC chlorophylls to the RC multimer, ultrafast charge separation (<500 fs) with a low yield starting from the singlet-excited `accessory' chlorophyll of the active branch, cation transfer from this `accessory' chlorophyll to a `special pair' chlorophyll and/or charge separation starting from this `special pair' chlorophyll (∼8 ps), and slow relaxation (∼50 ps) of the radical pair by conformational changes of the protein. The charge separation in the PS II RC can probably not be described as a simple trap-limited or diffusion-limited process, while for the PS II core and larger complexes the transfer of the excitation energy to the PS II RC may be rate limiting. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

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     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

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)     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Competition between annihilation and trapping leads to strongly reduced yields of photochemistry under ps-flash excitation

Excitation of photosynthetic systems with short intense flashes is known to lead to exciton-exciton annihilation processes. Here we quantify the effect of competition between annihilation and trapping for Photosystem II, Photosystem I (thylakoids from peas and membranes from the cyanobacterium Synechocystis sp.), as well as for the purple bacterium Rhodospirillum rubrum. In none of the cases it was possible to reach complete product saturation (i.e. closure of reaction centers) even with an excitation energy exceeding 10 hits per photosynthetic unit. The parameter introduced by Deprez et al. ((1990) Biochim. Biophys. Acta 1015: 295–303) describing the competition between exciton-exciton annihilation and trapping was calculated to range between 4.5 (PS II) and 6 (Rs. rubrum). The rate constants for bimolecular exciton-exciton annihilation ranged between (42 ps)^-1 and (2.5 ps)^-1 for PS II and PS I-membranes of Synechocystis, respectively. The data are interpreted in terms of hopping times (i.e. mean residence time of the excited state on a chromophore) according to random walk in isoenergetic antenna.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC II light harvesting complex II - P primary donor - PS I Photosystem I - PS II Photosystem II - PSU photosynthetic unit - RC reaction center     

Spectral hole burning of the primary electron donor state of Photosystem I

Persistent photochemical hole burned profiles are reported for the primary electron donor state P700 of the reaction center of PS I. The hole profiles at 1.6 K for a wide range of burn wavelengths (_B) are broad (FWHM310 cm^-1) and for the 45:1 enriched particles studied exhibit no sharp zero-phonon hole feature coincident with _B. The _B hole profiles are analyzed using the theory of Hayes et al. [J Phys Chem 1986, 90: 4928] for hole burning in the presence of arbitrarily strong linear electron-phonon coupling. A Huang-Rhys factor S in the range 4–6 and a corresponding mean phonon frequency in the range 35–50 cm^-1 together with an inhomogeneous line broadening of100 cm^-1 are found to provide good agreement with experiment. The zero-point level of P700^* is predicted to lie at710 nm at 1.6K with an absorption maximum at702 nm. The hole spectra are discussed in the context of the hole spectra for the primary electron donor states of PS II and purple bacteria.Abbreviations NPHB nonphotochemical hole burning - O.D. optical density - PSBH phonon sideband hole - PS I Photosystem I P680 - P700, P870, P960 the primary electron donors of Photosystem II, Photosystem I, Rhodobacter sphaeroides, Rhodopseudomonas viridis - PED primary electron donor - RC reaction center - ZPH zero-phonon holes     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation

Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc₃-Man₉-GlcNAc₂ from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.     

Binding affinity of bicarbonate and formate in herbicide-resistant D1 mutants of Synechococcus sp. PCC 7942

We examined the effects of mutations at amino acid residues S264 and F255 in the D1 protein on the binding affinity of the stimulatory anion bicarbonate and inhibitory anion formate in Photosystem II (PS II) in Synechococcus sp. PCC 7942. Measurements on the rates of oxygen evolution in the wild type and mutant cells in the presence of different concentrations of formate with a fixed bicarbonate concentration and vice versa, analyzed in terms of an equilibrium activator-inhibitor model, led to the conclusion that the equilibrium dissociation constant for bicarbonate is increased in the mutants, while that of the formate remains unchanged (11±0.5 mM). The hierarchy of the equilibrium dissociation constant for bicarbonate (highest to lowest, ±2 M) was: D1-F255L/S264A (46 M)>D1-F255Y/ S264A (31 M)D1-S264A (34 M)D1-F255Y (33 M)>wild type (25 M). The data suggest the importance of D1-S264 and D1-F255 in the bicarbonate binding niche. A possible involvement of bicarbonate and these two residues in the protonation of Q_B ^-, the reduced secondary plastoquinone of PS II, in the D1 protein is discussed.Abbreviations Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - MES 2-[N-morpholino]ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - Q_A bound plastoquinone, a one-electron acceptor in Photosystem II - Q_B another bound plastoquinone, a two-electron acceptor in Photosystem II This paper is dedicated to the memory of my dear friend Robin Hill-Govindjee.     

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     

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800–B850 gap from 750 cm^\s-1 at 0.1 MPa to 900 cm^-1 at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800–850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800–B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C₉-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (-0.4 cm⁰¹/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm⁰¹/MPa). The shift rate for B850 at 4.2 K is-0.28 cm^–1/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.Abbreviations B800 BChl antenna band absorbing (at room temperature) at 800 nm (B850, B875, B1015 are defined similarly) - CD circular dichroism - FC factor Franck-Condon factor - FMO comple Fenna-Matthews-Olson complex - L-S theory Laird-Skinner theory - LH1 core light-harvesting complex of the BChl antenna complexes - LH2 peripheral light-harvesting complex of the BChl antenna complexes - NPHB non-photochemical hole burning - P960 absorption band of special pair of Rhodopseudomonas viridis absorbing at 960 nm (room temperature). P870 of Rhodobacter sphaeroides is defined similarly - QM/MM results quantum mechanical/molecular mechanical results - RC reaction center - ZPH zero phonon hole     

2D NMR of paramagnetic metalloenzymes: Cyanide-inhibited horseradish peroxidase

Summary Two-dimensional (2D) proton NMR correlation spectroscopy, COSY, and nuclear Overhauser spectroscopy, NOESY, have been used to explore the applicability of these methods for the moderately large (42 KDa), paramagnetic cyanide-inhibited derivative of horseradish peroxidase, HRP-CN. The target resonances are those in the active site of HRP-CN which experience substantial hyperfine shifts and paramagnetic relaxation. The magnitude COSY experiment was found to yield cross peaks for all known spin-coupled heme substituents, as well as for the majority of non-heme hyperfine shifted protons, in spite of line widths of the order of 100 Hz. Moreover, the rapid relaxation of the hyperfine-shifted resonances allows the extremely rapid collection of useful 2D NMR data sets without the loss of information. For the heme, the combination of COSY cross peaks for the vinyl and propionate substituents, and NOESY cross peaks among these substituent protons and heme methyls, allows assignment of heme resonances without recourse to deuterium labeling of the heme. A seven-proton coupled spin system was identified in the upfield region that is consistent with originating from the proposed catalytic Arg³⁸ residue in the distal heme pocket, with orientation relative to the heme similar to that found in cytochromec peroxidase. The upfield hyperfine-shifted methyl group in the substrate binding pocket previously proposed to arise from Leu²³⁷ is shown to arise instead from an as yet unidentified Ile. NOESY spectra collected at very short (3 ms) and intermediate (20 ms) mixing times indicate that build-up curves can be obtained that should yield estimates of distances in the heme cavity. It is concluded that 2D NMR studies should be able to provide the heme assignments, aid in identifying the catalytic residues, and provide information on the spatial disposition of such residues in the active site for cyanide complexes of a number of intermediate to large paramagnetic heme peroxidases, as well as for other paramagnetic metalloenzymes with line widths of 100 Hz. Moreover, paramagnetic-induced hyperfine shifts and linewidths to 100 Hz need not interfere with the complete solution structure determination of a small paramagnetic protein solely on the basis of 2D NMR data.     

Cobalt induced changes in photosystem activity in Synechocystis PCC 6803: Alterations in energy distribution and stoichiometry

Adaptive responses to excess (supraoptimal) level of cobalt supplied to the growth medium were studied in the cyanobacterium Synechocystis PCC 6803. Growth of cells in the medium containing 10 M CoCl₂ led to a large stimulation (50%) in O₂-evolution and an overall increase (30%) in the photosynthetic electron transport rates. Analysis of variable Chl a fluorescence yield of PS II and immuno-detection of Photosystem II (PS II) reaction-center protein D1, showed a small increase (15–20%) in the number of PS II units in cobalt-grown cells. Cobalt-grown cells, therefore, had a slightly elevated PS II/PS I ratio compared to control.We observed alteration in the extent of energy distribution between the two photosystems in the eobalt grown cells. Energy was preferentially distributed in favour of PS II accompanied by a reduction in the extent of energy transfer from PS II to PS I in cobalt-grown cells. These cells also showed a smaller PS I absorption cross-section and a smaller size of intersystem electron pool than the control cells. Thus, our results suggest that supplementation of 10 M CoCl₂, to the normal growth medium causes multiple changes involving small increase in PS II to PS I ratio, enhanced funneling of energy to PS II and an increase in PS I electron transport, decrease PS I cross section and reduction in intersystem pool size. The cumulative effects of these alterations cause stimulation in electron transport and O₂ evolution.Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate - Chl a Chlorophyll a - Cyt blf Cytochrome blf - DCBQ 2,6-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DCPIP 2,6-dichlorophenol indophenol - DPC Diphenyl carbazide - F_o fluorescence when all reaction centers are open - F_M fluorescence yield when all reaction centers are closed - F_v variable chlorophyll fluorescence - HEPES N-2-hydroxyethyl piperazine-N'-2-ethanesulphonic acid - MV methyl viologen - NBT nitro-blue tetrazolium - pBQ para-benzoquinone - PB somes phycobilisomes - PC Phycocyanin - PQ plastoquinone - PS I Photosystem I - PS II Photosystem II - P700 reaction center Chl a of PS 1 - ST-and MT-flash single turnover and multiple turnover flash     

pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome (Cyt b ₅₅₉). At pH 6.5 it was found that Cyt b ₅₅₉ was reversibly photoreduced by red actinic light when Mn²⁺ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b ₅₅₉ was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942–10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b ₅₅₉ is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b ₅₅₉ increases, with increasing pH, the rate of irreversible photo-oxidation of -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680