Affinity and activity of non-native quinones at the QB site of bacterial photosynthetic reaction centers

Purple, photosynthetic reaction centers from Rhodobacter sphaeroides bacteria use ubiquinone (UQ₁₀) as both primary (Q_A) and secondary (Q_B) electron acceptors. Many quinones reconstitute Q_A function, while a few will act as Q_B. Nine quinones were tested for their ability to bind and reconstitute Q_A and Q_B functions. Only ubiquinone (UQ) reconstitutes both functions in the same protein. The affinities of the non-native quinones for the Q_B site were determined by a competitive inhibition assay. The affinities of benzoquinones, naphthoquinone (NQ), and 2-methyl-NQ for the Q_B site are 7 ± 3 times weaker than that at Q_A site. However, di-ortho-substituted NQs and anthraquinone bind tightly to the Q_A site (K _d ≤ 200 nM), and ≥1,000 times more weakly to the Q_B site, perhaps setting a limit on the size of the site. With a low-potential electron donor, 2-methyl, 3-dimethylamino-1,4-NQ, (Me-diMeAm-NQ) at Q_A, Q_B reduction is 260 meV, more favorable than with UQ as Q_A. Electron transfer from Me-diMeAm-NQ at the Q_A site to NQ at the Q_B site can be detected. In the Q_B site, the NQ semiquinone is estimated to be ≈60–100 meV higher in energy than the UQ semiquinone, while in the Q_A site, the semiquinone energy level is similar or lower with NQ than with UQ. Thus, the NQ semiquinone is more stable in the Q_A than in the Q_B site. In contrast, the native UQ semiquinone is ≈60 meV lower in energy in the Q_B than in the Q_A site, stabilizing forward electron transfer from Q_A to Q_B.     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q_B. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O₂ evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q_A and Q_B were found in the crystallized PSII. We propose that the extra quinones are located in the Q_B cavity and serve as a PSII intrinsic pool of electron acceptors.     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

Control of the maximal chlorophyll fluorescence yield by the Q<Subscript>B</Subscript> binding site

Differences in maximal yields of chlorophyll variable fluorescence (F_m) induced by single turnover (ST) and multiple turnover (MT) excitation are as great as 40%. Using mutants of Chlamydomonas reinhardtii we investigated potential mechanisms controlling F_m above and beyond the Q_A redox level. F_m was low when the Q_B binding site was occupied by PQ and high when the Q_B binding site was empty or occupied by a PSII herbicide. Furthermore, in mutants with impaired rates of plastoquinol reoxidation, F_m was reached rapidly during MT excitation. In PSII particles with no mobile PQ pool, F_m was virtually identical to that obtained in the presence of PSII herbicides. We have developed a model to account for the variations in maximal fluorescence yields based on the occupancy of the Q_B binding site. The model predicts that the variations in maximal fluorescence yields are caused by the capacity of secondary electron acceptors to reoxidize Q_A^–.     

Structural basis of cyanobacterial photosystem II Inhibition by the herbicide terbutryn

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q_B site in the D1 subunit and thus block the electron transfer from Q_A to Q_B. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q_B site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q_A, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.     

Action of tenuazonic acid,a natural phytotoxin,on photosystem II of spinach

Tenuazonic acid (TeA) is a putative phytotoxin obtained from Alternaria alternata, the organism that can cause brown leaf spot disease of Crofton weed (Eupatorium adenophorum). It is demonstrated here that the tenuazonic acid inhibits the activity of photosystem II (PSII); the I₅₀-value is 48 μg mL^?1. Evidences from chlorophyll fluorescence show that tenuazonic acid interrupts electron transport between Q_A and Q_B on the acceptor side of PSII. It does not have an effect on the antenna pigments, the oxygen-evolving complex (OEC) at the donor side of PSII. On the basis of the fluorescence induction kinetics and competition experiments with [¹⁴C]atrazine, it is shown that tenuazonic acid does not share the same binding environment with atrazine despite their common action target: the Q_B-site. It is concluded that tenuazonic acid is a member of a novel class of PSII inhibitors.     

Modeling the quinone-B binding site of the photosystem-II reaction center using notions of complementarity and contact-surface between atoms

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D1 and D2 photosystem-II reaction center proteins of cyanobacteria, algae, and plants. A model of the quinone (Q_B) binding site of the D1 protein is presented based upon the resolved structure of the Q_B binding pocket of the L subunit, and introducing novel quantitative notions of complementarity and contact surface between atoms. This model, built -without using traditional methods of molecular mechanics and restricted to residues in direct contact with Q_B, accounts for the experimentally derived functional state of mutants of the Dl protein in the region of Q_B. It predicts the binding of both the classical and phenol-type PSII herbicides and rationalizes the relative levels of tolerance of mutant phenotypes. © 1995 Wiley-Liss, Inc.     

Low-temperature modulation of the redox properties of the acceptor side of photosystem II: photoprotection through reaction centre quenching of excess energy

Although it has been well established that acclimation to low growth temperatures is strongly correlated with an increased proportion of reduced Q_A in all photosynthetic groups, the precise mechanism controlling the redox state of Q_A and its physiological significance in developing cold tolerance in photoautotrophs has not been fully elucidated. Our recent thermoluminescence (TL) measurements of the acceptor site of PSII have revealed that short‐term exposure of the cyanobacterium Synechococcus sp. PCC 7942 to cold stress, overwintering of Scots pine (Pinus sylvestris L.), and acclimation of Arabidopsis plants to low growth temperatures, all caused a substantial shift in the characteristic T_M of S₂Q_B^– recombination to lower temperatures. These changes were accompanied by much lower overall TL emission, restricted electron transfer between Q_A and Q_B, and in Arabidopsis by a shift of the S₂Q_A^–‐related peak to higher temperatures. The shifts in recombination temperatures are indicative of a lower activation energy for the S₂Q_B^– redox pair and a higher activation energy for the S₂Q_A^– redox pair. This results in an increase in the free‐energy gap between P680⁺Q_A^– and P680⁺Pheo^– and a narrowing of the free energy gap between Q_A and Q_B electron acceptors. We propose that these effects result in an increased population of reduced Q_A (Q_A^–), facilitating non‐radiative P680⁺Q_A^– radical pair recombination within the PSII reaction centre. The proposed reaction centre quenching could be an important protective mechanism in cyanobacteria in which antenna and zeaxanthin cycle‐dependent quenching are not present. In herbaceous plants, the enhanced capacity for dissipation of excess light energy via PSII reaction centre quenching following cold acclimation may complement their capacity for increased utilization of absorbed light through CO₂ assimilation and carbon metabolism. During overwintering of evergreens, when photosynthesis is inhibited, PSII reaction centre quenching may complement non‐photochemical quenching within the light‐harvesting antenna when zeaxanthin cycle‐dependent energy quenching is thermodynamically restricted by low temperatures. We suggest that PSII reaction centre quenching is a significant mechanism enabling cold‐acclimated organisms to acquire increased resistance to high light.     

Binding and release kinetics of inhibitors of Q−A oxidation in thylakoid membranes

Oxygen flash yield patterns of dark adapted thylakoid membranes as measured with a Joliot-type O₂-electrode indicate that inhibitors that block the oxidation of the reduced primary quinone Q^?_A of Photosystem II vary greatly in the rate of binding to and release from the inhibitor / Q_B binding environment. The ‘classical’ Photosystem-II herbicides like diuron and atrazine exhibit slow binding and release kinetics, whereas, for example, phenolic inhibitors, o-phenanthroline and synthetic quinones are exchanging quite rapidly with Q_B (about once per second or faster at inhibitor concentrations causing about 50% inhibition of O₂ evolution). No general relationship between the efficiency of the inhibitor and the exchange rate is observed; it depends mainly on the type of inhibitor. Based on the classical Kok model, equations are derived in order to calculate oxygen yields evolved by thylakoids in single-turnover flashes as a function of the rate constants of inhibitor binding to and release from the inhibitor / Q_B binding environment in the presence of an oxidized or semireduced Q_A · Q_B or Q_A · inhibitor complex. Fitting of theoretical and experimental values yields that o-phenanthroline binds much faster to an oxidized than to a semireduced Q_A · Q_B complex. This fits very well with the hypothesis that the Q^?_B affinity to the site is much higher than that of Q_B. In the case of i-dinoseb, however, inhibitor / quinone exchange seems to occur mainly in the semiquinone state. Possibilities to explain this result are discussed.     

Evidence for the Involvement of Loosely Bound Plastosemiquinones in Superoxide Anion Radical Production in Photosystem II

Recent evidence has indicated the presence of novel plastoquinone-binding sites, Q_C and Q_D, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O₂^•−) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O₂^•−, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the Q_B site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the Q_D site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b₅₅₉ (cyt b₅₅₉). Based on these results, we propose that O₂^•− is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the Q_B site and one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_C site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_D site seems to be ambiguous. In spite of the fact that the existence of Q_C and Q_D sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.     

Interaction of N,N,N′,N′-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the QB niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S₂Q_B⁻ and S₃Q_B⁻ (B-band), S₂Q_A⁻ (Q-band), and Y_D⁺Q_A⁻ (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y_n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q_B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q_B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q_B-site and to reduce the higher S-states of the Mn cluster.     

Effect of different methods of Ca<Superscript>2+</Superscript> extraction from PSII oxygen-evolving complex on the Q<Subscript>A</Subscript><Superscript>−</Superscript> oxidation kinetics

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn₄CaO₅ of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca²⁺ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca²⁺ extraction from OEC on the kinetics of the reduced primary electron acceptor (Q_A ^?) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from Q_A ^? to the secondary quinone acceptor Q_B. Electron transfer from Q_A ^? to Q_B in photosystem II membranes with an occupied Q_B site was slowed down by a factor of 8. However, addition of EGTA or CaCl₂ to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of Q_A ^? oxidation by Q_B in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca²⁺ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the Q_A ^? to Q_B electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca²⁺ depletion.     

Reorganization energies of the electron transfer reactions involving quinones in the reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In framework of the continuum electrostatics theory, the reorganization energies of the electron transfers Q_A^?–Q_B (fast phase), Bph^?–Q_A, P⁺–Q_A^?, and P⁺–Q_B^? in the photosynthetic bacterial reaction center have been calculated. The calculations were based on the static dielectric permittivity spatial distribution derived from the data on the electrogenesis, with the corresponding characteristic times relatively close to the reaction times of Q_A^?–Q_B (fast phase) and Bph^?–Q_A but much shorter than those times of the latter two recombination reactions. The calculated reorganization energies were reasonably close to the experimental estimates for Q_A^?–Q_B (fast phase) and Bph^?–Q_A but substantially lower than those of P⁺–Q_A^? and P⁺–Q_B^?. A higher effective dielectric permittivity contributes to this effect, but the dominant contribution is most probably made by a non-dielectric relaxation, especially for the P⁺–Q_B^? recombination influenced by the proton transfer. This situation calls for reconsidering of the current electron transfer rate estimates.     

EFFECT OF CHROMIUM(VI) ON PHOTOSYSTEM II ACTIVITY AND HETEROGENEITY OF SYNECHOCYSTIS SP. (CYANOPHYTA): STUDIED WITH IN VIVO CHLOROPHYLL FLUORESCENCE TESTS1

The inhibitory effect of Cr(VI) on the PSII of Synechocystis sp. was studied. Cr(VI) reduced O₂ evolution and inhibited the water‐splitting system in PSII. S‐states test and flash induction test showed that Cr(VI) exposure increased the proportion of inactivated PSII (PSII_X) and PSII_β reaction centers, which increased the fluxes of dissipated energy. JIP test and Q_A^? reoxidation test demonstrated that Cr(VI) treatment induces inhibition of electron transport from Q_A^? to Q_B/Q_B^? and accumulation of P₆₈₀⁺. More Q_A^? had to be oxidized through S₂(Q_AQ_B)^? charge recombination and oxidation by PQ9 molecules in PSII under Cr(VI) stress. These changes finally decreased the index of photosynthesis performance.     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B_A), bacteriopheopytin (H_A), the primary quinone (Q_A) to the secondary quinone (Q_B). Although the non-heme iron complex (Fe complex) is located between Q_A and Q_B, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q_A and Q_B in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q_A/B (E_m(Q_A/B)) and the Fe complex (E_m(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E_m(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E_m(Fe) downshift by 230 mV upon formation of Q_A⁻ (but not Q_B⁻) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Δ3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A₂ to decrease the PG content. Phospholipase A₂ treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q_A to the secondary quinone acceptor Q_B, (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A₂ treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A₂ brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q_B-binding site.     

Effects of Drought Stress on the Photosynthesis in Maize

To clarify how the components of the entire photosynthetic electron transport chain in response to drought stress in maize. The activities of photosystem II (PSII), photosystem I (PSI), and the electron transport chain between PSII and PSI of maize were investigated by prompt fluorescence (PF), delayed fluorescence (DF) and 820 nm modulated reflection (MR). Maize (Zea mays L.) plants were subjected to different levels of soil water availability including control, moderate and severe drought stress. A significant decrease in ?E₀, Ψ₀ and PI_ABS was found in maize treated with moderate drought stress. A significant increase in ABS/RC was observed, but there were no significant change in the fast MR phase and the amplitude of DF under moderate drought stress compared to the control. Under severe drought stress, the exchange capacity between Q_A to Q_B, reoxidation capacity of plastoquinol, and the oxidation and re-reduction rates of PC and P₇₀₀ all decreased. These results demonstrated that moderate drought stress reduced the photochemical activity of PSII from Q_A to PQH₂, while the photochemical activity of PSI was unscathed. However, severe drought stress inhibited the entire electron transport chain from the donor side of PSII to PSI-end electron acceptors. In addition, the photochemical activity of PSII is more sensitive to drought stress than PSI.     

Thermal phase and excitonic connectivity in fluorescence induction

Chl fluorescence induction (FI) was recorded in sunflower leaves pre-adapted to darkness or low preferentially PSI light, or inhibited by DCMU. For analysis the FI curves were plotted against the cumulative number of excitations quenched by PSII, n _q, calculated as the cumulative complementary area above the FI curve. In the +DCMU leaves n _q was <1 per PSII, suggesting pre-reduction of Q _A during the dark pre-exposure. A strongly sigmoidal FI curve was constructed by complementing (shifting) the recorded FI curves to n _q = 1 excitation per PSII. The full FI curve in +DCMU leaves was well fitted by a model assuming PSII antennae are excitonically connected in domains of four PSII. This result, obtained by gradually reducing Q _A in PSII with pre-blocked Q _B (by DCMU or PQH₂), differs from that obtained by gradually blocking the Q _B site (by increasing DCMU or PQH₂ level) in leaves during (quasi)steady-state e^? transport (Oja and Laisk, Photosynth Res 114, 15–28, 2012). Explanations are discussed. Donor side quenching was characterized by comparison of the total n _q in one and the same dark-adapted leaf, which apparently increased with increasing PFD during FI. An explanation for the donor side quenching is proposed, based on electron transfer from excited P680* to oxidized tyrosine Z (TyrZ^ox). At high PFDs the donor side quenching at the J inflection of FI is due mainly to photochemical quenching by TyrZ^ox. This quenching remains active for subsequent photons while TyrZ remains oxidized, following charge transfer to Q _A. During further induction this quenching disappears as soon as PQ and Q _A become reduced, charge separation becomes impossible and TyrZ is reduced by the water oxidizing complex.     

Application of fast chlorophyll a fluorescence kinetics to probe action target of 3-acetyl-5-isopropyltetramic acid

3-Acetyl-5-isopropyltetramic acid (3-AIPTA), an analogue of the phytotoxin tenuazonic acid, is a tetramic acid derivate. It is demonstrated here that 3-AIPTA is a multi-target inhibitor of root and shoot growth as well as photosynthesis. Based on fast chlorophyll fluorescence kinetics, 3-AIPTA blocks electron transport beyond Q_A on the acceptor side of PSII by competing with Q_B for the Q_B-binding site, but does not affect the donor side and the light harvesting function of the PSII antenna. Additionally, higher 3-AIPTA concentration also inhibits the reduction of end acceptors of PSI. Evidences from JIP-test and competitive replacement with [¹⁴C]atrazine showed that 3-AIPTA, like tenuazonic acid, does not share the same binding environment with the atrazine-like PSII inhibitors although they possess the common action target of the Q_B-site. It is deduced that tetramic acid families of natural products, where 3-AIPTA and tenuazonic acid belong to, is a novel inhibitor type of the photosynthetic electron transport chain.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.