EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Carotenoids as antioxidants: spin trapping EPR and optical study.

The role of several natural and synthetic carotenoids as scavengers of free radicals was studied in homogeneous solutions. A set of free radicals: *OH, *OOH, and *CH(3) were generated by using the Fenton reaction in dimethyl sulfoxide. It was shown that the spin trapping technique is more informative than optical methods for the experimental conditions under study. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) were used as spin traps for the EPR studies. The results show that the scavenging ability of the carotenoids towards radical *OOH correlates with their redox properties.     

Study on the photo-generation of superoxide radicals in Photosystem II with EPR spin trapping techniques

Direct EPR evidence of the photo-generation of superoxide radicals (O₂ ^–.) was obtained by using a novel spin trapping probe in spinach Photosystem II (PS II) membrane fragments. The production of O₂ ^–. was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) superoxide adducts (DEPMPO-OOH). The inhibition of O₂ ^–. formation by 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) and the 77 K fluorescence spectrum indicated that O₂ ^–. were generated from PS II, not from PS I. The inhibition of O₂ ^–. formation by DCMU also suggested that O₂ ^–. were generated from the Q_Bbinding site, not at a site prior to DCMU blockage. The extrinsic proteins and Mn are very important to eliminate O₂ ^–., showing that the oxygen-evolving system is involved in O₂ ^–. removal rather than production.This revised version was published online in October 2005 with corrections to the Cover Date.     

Location and dynamics of alamethicin in unilamellar vesicles and thylakoids as model systems. A spin label study

Location and dynamics of the voltage-dependent pore-forming icosapeptide alamethicin have been studied using spin labels which were linked directly and via spacers to the C-terminus of the amphiphilic alpha-helix. Ion-transport activities of these derivatives were found to be very similar to those of natural alamethicin in green plant thylakoids chosen as a model system. The shape of the electron spin resonance spectra indicates segmental motion of the nitroxide rather than rotation of the whole peptide. A population of spins showing narrow lines in the presence of thylakoids or lipid vesicles is attributed to alamethicin in the aqueous solution. A second population shows rotational correlation times greater than 10(-9) s and is bound to the membranes, the C-termini residing in an environment with a polarity close to that of water. This population is inaccessible to the hydrophilic, charged line broadening agent chromium oxalate. Since spectral shapes and amplitudes of spectra are unchanged by additions of unlabelled peptide, it is concluded that the ESR detectable spins are bound to peptides essentially in the monomeric state. Alamethicin induced pore formation under flash illumination is demonstrated by measurement of kinetics of proton deposition in the thylakoid interior. When pores are opened by illuminating thylakoids and thus applying a membrane potential, mainly the bound population is affected by a process reversibly suppressing the signal, whereas only limited disappearance of label from the external medium is detected. Apparently, the potential causes a change in the conformation of the peptide which leads to a further immobilisation of the label, possibly due to a deeper insertion of the alpha-helices into the lipid membrane. However, evidence has been presented experimentally that there is no detectable change of potential prior to the opening of the pore.     

A thermoluminescence study of the effects of nitrite on photosystem II in spinach thylakoids.

The site of action of nitrite on PS II was investigated by measuring the TL profile of nitrite-treated spinach thylakoid membranes. Three bands were observed in control, which were identified as the Q band (7 degrees C), the B band (24 degrees C) and the C band (57 degrees C). In the presence of 20 mmol/L nitrite, the intensity of the Q band decreased, the B band upshifted to 46 degrees C but the C band disappeared. The suppression of the Q band and the upshift of the B band suggested that nitrite caused inhibition at the water oxidizing complex. The effects of nitrite also remained the same in the presence of chloride. In case of ion-sufficient thylakoid membranes, nitrite decreased the Q band peak intensity and caused an upshift in the B band peak temperature. Nitrite showed similar effects in the presence of DCMU. This suggested that the site of action of nitrite is not at the acceptor side but at the donor side of PS II. The inhibition shown by nitrite has been found to be specific for nitrite anion. No other anions such as formate, fluoride or nitrate, were effective.     

An EPR study of Signal II in oriented photosystem II membranes

Signal II of plant photosynthesis, which is thought to be due to a plastosemiquinone cation radical, has been studied by EPR at 9 and 35 GHz in non-oriented and partly oriented PS II particles. The spectra measured of the oriented particles at 35 GHz show that the molecular Z-axis, which is the axis perpendicular to the plane of the radical, makes an angle of 60° with the membrane normal. All spectra could be computer-simulated with one set of parameters. This set is essentially the same as that given earlier on the basis of EPR spectroscopy on non-oriented membranes [(1985) Biochim. Biophys. Acta 809, 421-428], except that the bond bending of the hydroxyl group on ring position 1 is found to be 60°, resulting in a somewhat smaller isotropie hyperfine splitting of the hydroxyl proton.Signal IIEPROrientationHyperfine coupling     

Proton release from water oxidation by photosystem II: similar stoichiometries are stabilized in thylakoids and PSII core particles by glycerol

During the four-stepped catalytic cycle of water oxidation by photosystem II (PSII) molecular oxygen is released in only one of the four reaction steps whereas the release of four protons is distributed over all steps. In principle, the pattern of proton production could be taken as indicative of the partial reactions with bound water. In thylakoids the extent and rate of proton release varies as function of the redox transition and of the pH without concomitant variations of the redox pattern. The variation has allowed to discriminate between deprotonation events of peripheral amino acids (Bohr effects) as opposed to the chemical deprotonation of a particular redox cofactor, and of water. In contrast, in thylakoids grown under intermittent light, as well as in PSII core particles the pattern of proton release is flat and independent of the pH. This has been attributed to the lack in these materials of the chlorophyll a,b-binding (CAB) proteins. We now found that a thylakoid-like, oscillatory pattern of proton release was restored simply by the addition of glycerol which modifies the protein–protein interaction. Being a further proof for the electrostatic origin of the greater portion of proton release, this effect will serve as an important tool in further studies of water oxidation.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH.) and singlet oxygen ((1)O2), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 degrees C). Production of OH., evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH. production. These observations reveal that OH. is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH., whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH. is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH. scavengers. This finding indicates that the production of these two species was independent and that the production of (1)O2 is not closely linked to PSII donor side.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH) and singlet oxygen (¹O₂), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 °C). Production of OH, evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH production. These observations reveal that OH is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH, whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH scavengers. This finding indicates that the production of these two species was independent and that the production of ¹O₂ is not closely linked to PSII donor side.     

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (<15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.     

Limited sensitivity of pigment photo-oxidation in isolated thylakoids to singlet excited state quenching in photosystem II antenna

Light-induced pigment oxidation and its relation to excited state quenching in photosystems antennae have been investigated in isolated thylakoids. The results indicate that (i) chlorophyll oxidation takes place in two sequential steps. A slow initial phase is followed by a steep increase in the bleaching rate when more than one quarter of the chromophores are oxidised. (ii) During the initial slow phase, the carotenoid pool is bleached with an apparent rate which is about three times faster than that found for chlorophyll a and more than six times faster than that of chlorophyll b. (iii) Pigment bleaching has been observed both in photosystem I and photosystem II, and it has been possible to estimate a similar carotenoid bleaching rate in the two photosystems. (iv) The protection conferred by singlet state quenchers in the initial slow phase of pigment oxidation is modest. Taking into consideration that both the photosystems are subjected to the oxidative treatment, a somewhat larger protective effect than those estimated for photo-inhibition in thylakoids [S. Santabarbara, F.M. Garlaschi, G. Zucchelli, R.C. Jennings, Biochim. Biophys. Acta 1409 (1999) 165-170] can be computed, although it is less than 50% of the expected level on the basis of the observed reciprocity to the number of incident photons. (v) Pigment oxidation is associated with the loss of membrane ultra-structure, which is interpreted as originating from a decrease in grana stacking. The dynamics of loss of membrane ultra-structure parallel the phases observed for chlorophyll photo-bleaching.     

Detection of nitric oxide in tissue by spin trapping EPR spectroscopy and triacetylglycerol extraction

A modified method based on EPR spin trapping and triacetylglycerol extraction was used for tissue nitric oxide (NO) detection at room temperature. NO signal intensity was stable for about 1.5 h and the detection limit of this method was less than 200 pmol g^–1 tissue. Using this method, we report evidence that NO production in vivo can be inhibited by adriamycin in mice livers.     

A three-state model for energy trapping and chlorophyll fluorescence in photosystem II incorporating radical pair recombination

The multiphasic fluorescence induction kinetics upon a high intensity light pulse have been measured and analyzed at a time resolution of 10 micros in intact leaves of Peperomia metallica and Chenopodium album and in chloroplasts isolated from the latter. Current theories and models on the relation between chlorophyll fluorescence yield and primary photochemistry in photosystem II (PSII) are inadequate to describe changes in the initial phase of fluorescence induction and in the dark fluorescence level F(0) caused by pre-energization of the system with single turnover excitation(s). A novel model is presented, which gives a quantitative relation between the efficiencies of primary photochemistry, energy trapping, and radical pair recombination in PSII. The model takes into account that at least two turnovers are required for stationary closure of a reaction center. An open reaction center is transferred with high efficiency into its semiclosed (-open) state. This state is characterized by Q(A) and P680 in the fully reduced state and a lifetime equal to the inverse of the rate constant of Q(A)(-) oxidation (approx. 250 micros). The fluorescence yield of the system with 100% of the centers in the semiclosed state is 50% of the maximal yield with all centers in the closed state at fluorescence level F(m). A situation with approximately 100% of the centers in the semiclosed state is reached after a single turnover excitation in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The lifetime of this state under these conditions is approximately 10 s. Closure of a semiclosed (-open) center occurs with low efficiency in a second turnover. The low(er) efficiency is caused by the rate of P(+) reduction by the secondary donor Y(Z) being competitive with the rate of radical pair recombination in second and following turnovers. The single-turnover-induced alterations in the initial kinetics of the fluorescence concomitantly with a 15-25% increase in F(o) can be simulated with the present so called three-state model of energy trapping. The experimental data suggest evidence for an electrostatic effect of local charges in the vicinity of the reaction center affecting the rate of radical pair recombination in the reaction center.     

Oxidation of biological electron donors and antioxidants by a reactive lactoperoxidase metabolite from nitrite (NO2-): an EPR and spin trapping study

We report that a lactoperoxidase (LPO) metabolite derived from nitrite (NO2-) catalyses one-electron oxidation of biological electron donors and antioxidants such as NADH, NADPH, cysteine, glutathione, ascorbate, and Trolox C. The radical products of the reaction have been detected and identified using either direct EPR or EPR combined with spin trapping. While LPO/H2O2 alone generated only minute amounts of radicals from these compounds, the yield of radicals increased sharply when nitrite was also present. In aerated buffer (pH 7) the nitrite-dependent oxidation of NAD(P)H by LPO/H2O2 produced superoxide radical, O2*-, which was detected as a DMPO/*O2H adduct. We propose that in the LPO/H2O2/NO2-/biological electron donor systems the nitrite functions as a catalyst because of its preferential oxidation by LPO to a strongly oxidizing metabolite, most likely a nitrogen dioxide radical *NO2, which then reacts with the biological substrates more efficiently than does LPO/H2O2 alone. Because both nitrite and peroxidase enzymes are ubiquitous our observations point at a possible mechanism through which nitrite might exert its biological and cytotoxic action in vivo, and identify some of the physiological targets which might be affected by the peroxidase/H2O2/nitrite systems.     

Stimulation and inhibition of photosystem II electron transport in cyanobacteria by ions interacting with the cytoplasmic face of thylakoids

The mechanism by which suspension medium ions regulate the rate of photoinduced electron transport across photosystem II was investigated with ion permeabilized cells of the cyanobacterium Anacystis nidulans. Electron transport was measured as the reduction of the electroneutral acceptor dichlorophenol indophenol, whose surface concentration is independent of electrostatic membrane potential. Potassium salts stimulate photoinduced electron transport at low concentrations and inhibit it at higher concentrations. No inhibition is observed when an antichaotropic anion is associated with potassium, while the inhibition is more severe the stronger the chaotropic character of the anion. Neutralization of the surface charge by potassium ions ligated to negatively charged membrane sites at the cytoplasmic side is a prerequisite for the expression of the chaotropic inhibition of photosystem II electron transport.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPC 1,5-diphenyl carbazide - FeCN ferricyanide anion - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - PS photosystem - TEC³⁺ tris ethylene diamine cobalt cation     

EPR spin trapping of a radical intermediate in the urate oxidase reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5-diethoxyphosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.     

Tyrosine radicals in photosystem II and related model compounds. Characterization by isotopic labeling and EPR spectroscopy

Deuteration at selected positions on the phenol ring and at the beta-methylene carbon for the YD.tyrosine radical in Photosystem II in the cyanobacterium Synechocystis 6803 was achieved by growing the organism under conditions in which it is a functional aromatic amino acid auxotroph (Barry, B. A., and Babcock, G. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7099-7103). A series of model tyrosine radicals, also deuterated at specific sites on the aromatic ring and the methylene group, was generated by UV irradiation of frozen solutions. The EPR spectra of the specifically deuterated in vivo radicals confirm that YD.is a tyrosine; nevertheless its spectra differ from those of the tyrosine models. By comparing the EPR spectra of the specifically deuterated radicals with those of the fully protonated, the hyperfine couplings of the various protons of both YD.and the model compound radicals were determined. For both species, the unpaired electron spin density distribution is essentially identical and follows an odd-alternant pattern with high rho values at the carbons ortho and para to the tyrosine phenol oxygen; the meta positions have low spin densities. The differences in EPR spectral characteristics for the two radicals are rationalized as arising from variations in the conformation of the beta-methylene group with respect to the phenol head group. Considering these EPR results and those reported for other model and naturally occurring tyrosine radicals, we conclude that this situation is general; there is little deviation in this class of compounds from the odd-alternant spin density distribution; variations in EPR lineshapes arise primarily from variations in beta-methylene orientation. The conformation of the -CH2- group in biologically active tyrosine radicals deviates from that observed in the models and may be functionally significant. Because the EPR spectrum of YZ., the second redox active tyrosine radical in Photosystem II, is identical to that of YD., we conclude that the two radicals are in similar protein environments, a conclusion that is supported by the protein sequences in the vicinity of the two radicals.     

Intactness of the oxygen-evolving system in thylakoids and Photosystem II particles

Photosystem II activity of oxygen-evolving membranes can be quantified by their capacity to do charge separation or their capacity to transport electrons. In this study using flash excitation of saturating intensity, charge separation is measured by absorption changes in the ultraviolet region of the spectra associated with primary-quinone reduction, and electron transport is measured by oxygen flash yield. These methods are applied to thylakoids and three different types of Photosystem II particles. In thylakoids electron-transport activity is 75–85% of charge separation activity. In Photosystem II particles this percentage is 60–70%, except for the BBY type (Berthold, D.A., Babcock, G.T. and Yocum, C.F. (1981) FEBS Lett. 135, 231–234), in which it is only 29%. These estimates of non-functional oxygen-evolving centers agree within experimental error, except for the BBY particle, with the quantum requirement for oxygen evolution measured under light-limited conditions. These reaction centers that are non-functional in oxygen evolution occur during sample preparation and are not a result of inhibition by ferricyanide or quinone acceptor systems. In thylakoids on the first flash, absorption changes at 325 nm do not show significant contributions from oxygen evolution S-state transitions. In the presence of ferricyanide the absorption change at 325 nm does have a significant contribution from Q₄₀₀ in thylakoids, but considerably less in Photosystem II particles.