Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

Fluorescence nanoscopy by ground-state depletion and single-molecule return

We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein-labeled (living) samples, proving a simple yet powerful super-resolution approach.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Light-dependent fluorescence variations in intact leaves

Transient variations in the fluorescence from intact Phytolaccaamericana leaves after the onset of illumination were measuredunder various light and dark conditions. Dark-adapted leaveswhen illuminated with strong light underwent an intensity variationwith a peak; the fluorescence intensity reaching its peak severalseconds after the onset of illumination then decreasing to asteady level. The peak height relative to the steady level increasedwith the increasing intensity of actinic light. Pre-illuminationof the dark-adapted leaves with strong light caused a markedlowering of the peak. About 20 min of dark incubation was requiredfor the light-adapted leaves to return to the dark-adapted state.All of the action spectra, for the peak, the steady level andthe effect of light in post-illumination to inhibit recoveryto the dark state, showed high bands due to chlorophyll b andcarotenoid absorption and low bands due to chlorophyll a absorption.We concluded that the light absorbed by photosystem 2 is responsiblefor these phenomena. (Received April 21, 1975; )     

Quenching of triplet state fluorophores for studying diffusion-mediated reactions in lipid membranes

An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane-associated electron spin resonance labels on the triplet state kinetics of membrane-bound fluorophores. Singlet-triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied, when free in aqueous solutions, with LRB bound to a lipid in a liposome, and in the presence of different local concentrations of the electron spin resonance label TEMPO. By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy, a strong fluorescence signal can be combined with the ability to monitor low-frequency molecular interactions, at timescales much longer than the fluorescence lifetimes. Both in solution and in membranes, the measured relative changes in the singlet-triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules. These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected. The proposed strategy is broadly applicable, in terms of possible read-out means, types of molecular interactions that can be followed, and in what environments these interactions can be measured.     

Oxidation and reduction of plastoquinone by photosynthetic and respiratory electron transport in a cyanobacterium Synechococcus sp.

The I-D dip, an early transient of the fluorescence induction, was examined as a means to monitor redox changes of plastoquinone in cells of a cyanobacterium, Synechococcus sp. That the occurrence of the dip depends upon the reduced state of the plastoquinone pool was indicated by observations that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not affect the initial rise to I but abolished the subsequent decline from I to D and that illumination of the cells with light 1, prior to fluorescence measurements, eliminated the transient. The I-D dip was prominent in freshly harvested cells containing abundant endogenous substrates, disappeared slowly as the cells were starved by aeration but reappeared on addition of fructose to the starved cells in the dark. The dip that had been induced by a brief illumination of the starved cells with light 2 was rapidly diminished in the dark and KCN inhibited the dark decay of the transient. The results indicate that plastoquinone is reduced with endogenous as well as exogenous substrates and oxidized by a KCN-sensitive oxidase in the dark, thus providing strong support for the view that plastoquinone of photosynthetic electron transport also functions in respiration. In addition, the occurrence of a cyclic pathway of electrons from Photosystem I to plastoquinone, possibly via ferredoxin or NADP, was suggested. Several lines of evidence indicate that, under a strong light 2, Photosystem I-dependent oxidation of plastoquinone predominates over Photosystem II-dependent reduction of the quinone in the cyanobacterium which contains Photosystem I more abundantly than Photosystem II.     

Photochemistry in the isolated Photosystem II reaction-centre core complex.

The photochemistry of the isolated Photosystem II reaction-centre core from pea and the green alga Scenedesmus was examined by e.s.r. Two types of triplet spectrum were observed in addition to the spin-polarized reaction-centre triplet previously identified. The additional triplet formed on continuous illumination at 4.2 K was attributed to a monomeric phaeophytin molecule. The second triplet, which was stable in the dark at 4.2 K following illumination, was assigned to the radical pair Donor+I-. This provides evidence that an electron donor to chlorophyll P680 is present on the polypeptide D1-polypeptide D2-cytochrome b-559 core complex.     

Molecular basis of photochromism of a fluorescent protein revealed by direct <Superscript>13</Superscript>C detection under laser illumination

Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of ¹³C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between C^α and C^β of Y63 in the chromophore, the β-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of β-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.     

Nonphotochemical Reduction of the Plastoquinone Pool in Sunflower Leaves Originates from Chlororespiration

We investigated the relationship between nonphotochemical plastoquinone reduction and chlororespiration in leaves of growth-chamber-grown sunflower (Helianthus annuus L.). Following a short induction period, leaves of previously illuminated sunflower showed a substantially increased level of minimal fluorescence following a light-to-dark transition. This increase in minimal fluorescence was reversed by far-red illumination, inhibited by rotenone or photooxidative methyl viologen treatment, and stimulated by fumigation with CO. Using flash-induced electrochromic absorption-change measurements, we observed that the capacity of sunflower to reduce plastoquinone in the dark influenced the activation state of the chloroplast ATP synthase, although chlororespiratory transmembrane electrochemical potential formation alone does not fully explain our observations. We have added several important new observations to the work of others, forming, to our knowledge, the first strong experimental evidence that chlororespiratory, nonphotochemical plastoquinone reduction and plastoquinol oxidation occur in the chloroplasts of higher plants. We have introduced procedures for monitoring and manipulating chlorores-piratory activity in leaves that will be important in subsequent work aimed at defining the pathway and function of this dark electron flux in higher plant chloroplasts.     

The phot LOV2 domain and its interaction with LOV1

Phot proteins are homologs of the blue-light receptor phototropin. We report a comparative study of the photocycles of the isolated, light-sensitive domains LOV1 and LOV2 from Chlamydomonas reinhardtii phot protein, as well as the construct LOV1/2 containing both domains. Transient absorption measurements revealed a short lifetime of the LOV2-wt triplet state (500 ns), but a long lifetime (287 micros) of the triplet in the mutant LOV2-C250S, in which the reactive cysteine is replaced by serine. For LOV1, in comparison, corresponding numbers of 800 ns and 4 micros for the two conformers in LOV1-wt, and 27 micros for LOV1-C57S have been reported. The triplet decay kinetics in the mixed domains LOV1/2-wt, LOV1/2-C57S, and LOV1/2-C250S can be analyzed as the superposition of the behavior of the corresponding single domains. The situation is different for the slow, thermal reaction of the photoadduct back to the dark form. Whereas the individual domains LOV1 and LOV2 show two decay components, the double domains LOV1/2-C57S and LOV1/2-C250S both show only a single component. The interaction of the two domains does therefore not manifest itself during the lifetime of the triplet states, but changes the decay behavior of the adduct states.     

Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Changes in the distribution of light energy between the two photosystems in spinach leaves

Time courses of chlorophyll fluorescence at room temperature and fluorescence spectra at 77 K were measured to investigate the light-induced changes in the distribution of light energy between the two photosy stems in young spinach leaves. Illumination of the dark adapted leaves with primarily system II light induced typical fluorescence transients at room temperature. Fluorescence spectra at 77 K showed that the intensity of system II fluorescence at 77 K changed nearly in parallel with the fluorescence transients at room temperature within the range from M₁ to T during illumination of the leaf. Illumination of the dark adapted leaves with light I produced an increase of system II fluorescence measured at 77 K. The characteristics of the changes induced by light I or II were different, showing that these two effects are related to different mechanisms. These results suggest that the dark state in spinach leaves is state II, that light I induces a state II to I transition, while light II induces fluorescence changes that are produced by mechanisms other than state I-state II transitions.     

Bacteriochlorophyll fluorescence of purple bacteria at low redox potentials. The relationship between reaction center triplet yield and the emission yield.

This work describes fluorescence yield measurements in suspensions of strains of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in which the iron . quinone complex (X) was chemically reduced (state [PIX-]; P is the reaction center bacteriochlorophyll dimer, I is the long wavelength bacteriopheophytin), and compares these with the fluorescence observed when all the traps are open (state [PIX]) and with the fluorescence observed when all the traps are closed (state [P+IX]). At 77 K the amplitude and the shape of the fluorescence emission spectrum in [PIX-] are identical to those observed in [PIX]. This is a strong indication that all the extra fluorescence observed at room temperature in [PIX-] is, in fact, caused by an efficient back reaction [P+I-X-] leads to [P*IX-]. Using an equation similar to the original Vredenberg-Duysens relationship (Vredenburg, W.J. and Duysens, L.N.M. (1963) Nature 197, 355-357) but now assuming that a single reaction center has a probability pt of trapping an excitation and (1--pt) of re-emitting it to the surroundings, we are able to calculate pt as a function of the temperature by measuring the fluorescence in [PIX], [PIX-] and [P+IX] as a function of the temperature. The calculated pt values agree reasonably well with triplet yields measured in isolated reaction centers. Finally, we have measured the reaction center triplet yield (PTR) in intact systems and we have shown that the sum of the triplet yield and the remaining loss processes (PL) in the antenna bacteriochlorophyll including the bacteriochlorophyll dimer (such as fluorescence, internal conversion or direct triplet formation) is approximately constant; if we assume that at 77 K the only process which occurs in the reaction center is the formation of a reaction center triplet, than PTR + PL=1. The energy barrier between [P*IX-] and [P+I-X-] was estimated to be 0.11--0.15 eV for a set of preparations.     

Light‐independent thermoluminescence from thylakoids of greening barley leaves. Evidence for involvement of oxygen radicals and free chlorophyll

A study was conducted to identify biophysical markers which change in response to changing chlorophyll organization during plant development. When heated to around 70°C in the dark, barley thylakoids emit red thermoluminescence (TL). This is a pure chemiluminescence signal and distinct from the lower-temperature TL bands of thylakoids that are seen only with preillumination. The development of the light-independent, 70°C TL band was investigated following transfer of dark-grown barley leaves to the light. Because of the rapidly increasing chlorophyll content of the plastid membrane, the TL signal was normalized against either chlorophyll or tissue mass of the starting material. In either case, the extent of the TL signal reached a maximum in the early hours of greening and then declined. The drop in signal over 20 h was approximately 50% for TL per unit tissue mass, and well over 90% for TL per unit chlorophyll. Exposure of plastid membrane samples to hydrogen peroxide for several minutes caused a large increase in light-independent TL, while addition of ascorbate caused substantial quenching. The fluorescence profiles of dark-grown barley leaves were recorded following transfer to the light. Basal fluorescence (F₀) reaches a substantial level after just seconds of illumination. Over the next few hours, F₀ increases only slightly and then starts to decline. The decline in F₀ is correlated with an increase in variable fluorescence (F_v) which indicates the appearance of active photosystem II. It is concluded that the early peak in F₀ reflects a state in which the leaves contain a maximum amount of disorganized chlorophyll. Considering the TL and fluorescence data together, we propose the following: When chlorophyll first appears in the system, it is not properly assembled into the complexes that offer photochemical or non-photochemical quenching of the excited state. Thus, fluorescence and parallel chlorophyll triplet formation are prevalent. The triplets cause generation of active oxygen resulting in lipid peroxidation and/or other radical-generating processes. When the membranes are heated, increased interaction of the radicals with chlorophyll generates chemiluminescence. We thus conclude that light-independent thermoluminescence is a marker for actual damage arising from poor chlorophyll organization and propose that this parameter might be usefully applied for assessing stress effects.     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q(A)) of photosystem (PS) II. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 micros to 5 s). The about 20% lowering of the maximum fluorescence yield F(M), observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH(2) by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH(2) starts getting reoxidized by PS I activity. NAD(P)H-dependent restoration of F(M) was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl(2) that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F(M). Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F(0) allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F(0) level (Q(0)) and to compare it with the fractional quenching at the F(M) level (Q(M)). The experimentally determined Q(0)/Q(M) ratios were found to be equal to the corresponding F(0)/F(M) ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Electron transfer from the water oxidizing complex at cryogenic temperatures: the S1 to S2 step

We report the detection of a "split" electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S(1) state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa*- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the "S1 split signal", which we provisionally assign to S1X*, where X could be YZ* or Car*+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40-50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).     

Effect of riboflavin and light on the secondary structure of DNA

S1 nuclease hydrolysis and benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography have been used to study the effect of riboflavin and visible light on DNA. Native calf thymus DNA was incubated with riboflavin in the presence of fluorescent light for various time periods and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Incubation with riboflavin and illumination caused adherence to BND-cellulose indicating the production of single stranded regions or breaks in the native double stranded molecules. However, when incubation was done in dark and in the presence of triplet excited state quencher, potassium iodide, a reduced adherence of DNA to BND-cellulose was seen. Plasmid pBR322 DNA was also treated with riboflavin under these conditions and subjected to agarose gel electrophoresis. No degradation could be seen in dark incubated and potassium iodide treated samples. These results indicate that the adherence of DNA to BND-cellulose in dark is possibly due to the binding of aromatic residues to the resin suggesting the formation of a complex between riboflavin and DNA.     

Effects of anaerobiosis as probed by the polyphasic chlorophyll a fluorescence rise kinetic in pea (Pisum sativum L.)

We analysed the changes of the chlorophyll (Chl)a fluorescence rise kinetic (from 50 s to 1 s) that occur when leaves or chloroplasts of pea ( Pisum sativum L.) are incubated under anaerobic conditions in the dark. In control leaves, Chl a fluorescence followed a typical O-J-I-P polyphasic rise [Strasser et al. (1995) Photochem Photobiol 61: 32–42]. Anaerobiosis modified the shape of the transient with the main effect being a time-dependent increase in the fluorescence yield at the J-step (2 ms). Upon prolongation of the anaerobic treatment (> 60 min), the O-J-I-P fluorescence rise was eventually transformed to an O-J (J = P) rise. A similar transformation was observed when pea leaves were treated with DCMU or sodium dithionite. Anaerobiosis resulted in a 10–20% reduction in the maximum quantum yield of the primary photochemistry of Photosystem II, as measured by the ratio of the maximal values of variable and total fluorescence (F_V/F_M). When the leaves were returned to the air in the dark, the shape of the fluorescence transient showed a time-dependent recovery from the anaerobiosis-induced change. The original O-J-I-P shape could also be restored by illuminating the anaerobically treated samples with far-red light but not with blue or white light. Osmotically broken chloroplasts displayed under anaerobic conditions fluorescence transients similar to those observed in anaerobically treated leaves, but only when they were incubated in a medium comprising reduced pyridine nucleotides (NADPH or NADH). As in intact leaves, illumination of the anaerobically treated chloroplasts by far-red light restored the original O-J-I-P transient, although only in the presence of methyl viologen. The results provide additional evidence for the existence of a chlororespiratory pathway in higher plant cells. Furthermore, they suggest that the J-level of the fluorescence transient is strongly determined by the redox state of the electron carriers at the PS II acceptor side.     

Triplet state EPR of reaction centers from the HisL173→LeuL173 mutant of Rhodobacter sphaeroides which contains a heterodimer primary donor

Electron paramagnetic resonance (EPR) spectroscopy has been used to examine the triplet states in reaction centers of Rhodobacter sphaeroides which have undergone a genetic modification affecting the primary donor. Reaction centers containing the His^L173Leu^L173 substitution in the amino acid sequence have a primary donor which consists of a BChl-BPh heterodimer. The triplets formed in this heterodimer reaction center were compared with those formed in the wild-type reaction center which contains the BChl-BChl homodimer. Both reaction centers transfer triplet energy to the carotenoid under illumination at liquid nitrogen temperatures (90 K). However, the intensity of the carotenoid triplet signal is significantly decreased in the Leu^L173 mutant compared with the wild-type reaction center. At 12 K, in wild-type reaction centers only the primary donor triplet is observed. The Leu^L173 mutant exhibits a signal similar to that observed by Bylina et al. (1990) in His^M200Leu^M200 mutant reaction centers from Rb. capsulatus. The values of the zero-field splitting parameters of this triplet are discussed within the context of various models for the primary donor triplet state. No alteration in the ability of the carotenoid to quench the primary donor triplet state results from mutations at these sites.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin - EPR electron paramagnetic resonance - LDAO lauryl-dimethylamine N-oxide