Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

Slow fluorescence quenching of Type A chloroplasts. Resolution into two components

The divalent-cation-specific ionophore A23187 is used to define two components of the slow fluorescence quenching of type a spinach chloroplasts: ionophore-reversible and ionophore-resistant quenching. Ionophore-reversible quenching predominates at relatively low light intensities and approaches saturation as light levels are increased. It is sensitive to uncouplers and to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and is dark reversible. At high light intensities the bulk (> 80%) of slow fluorescence quenching is ionophore-resistant. Ionophore-resistant quenching is stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at pH 7.6 and by both CCCP and methylamine at pH 9.0. It is insensitive to DCMU and is not reversed in subsequent darkness. Taken together, the two components account for all quenching observed in Type A chloroplasts.Ionophore-reversible quenching is identified with the Mg²⁺-mediated fluorescence quenching described by Krause (Biochim. Biophys. Acta (1974) 333, 301–313) and by Barber and Telfer (in Membrane Transport in Plants (Dainty, J., and Zimmermann, U., eds.), pp. 281–288, Springer-Verlag, Berlin, 1974). Ionophore-resistant quenching, a first-order process requiring high light, resembles the quenching reported by Jennings et al. (Biochim. Biophys. Acta (1976) 423, 264–274).The resolution of the fluorescence quenching phenomenon into two distinct components reconciles the apparently contradictory observations of these earlier investigations.     

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine and human serum albumin

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine (CECT) and human serum albumin (HSA) were investigated using fluorescence, UV absorption spectroscopic and molecular modeling techniques under simulative physiological conditions. The intrinsic fluorescence intensity of HSA was decreased with the addition of CECT. The fluorescence data handled by Stern–Volmer equation proved that the quenching mechanism of the interaction between CECT and HSA was a static quenching procedure. The binding constants evaluated utilizing the Lineweaver–Burk equation at 17, 27 and 37?°C, were 2.340?×?10⁴, 2.093?×?10⁴ and 1.899?×?10⁴?L?mol^?1, respectively. The thermodynamic parameters were calculated according to van’t Hoff equations. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic force played a major role in the binding process of CECT to HSA, which was consistent with the results of the molecular modeling study. In addition, the effect of other ions on the binding constant of CECT-HSA was examined.     

The penetration of local anesthetics into the red blood cell membrane as studied by fluorescence quenching.

The local anesthetics procaine and tetracaine were found to quench the fluorescence of the probes N-octadecyl naphthyl-2-amine 6-sulfonic acid and 12-(9-anthroyl)stearic acid in the presence of erythrocyte membranes. This quenching was shown to be due to the aromatic amine of the procaine and tetracaine molecules. Lidocaine, an active anesthetic that does not contain an aromatic amine in the same position as does procaine and tetracaine did not quench either of the fluorophores. The preferential quenching of the fluorescent probes by procaine and tetracaine indicated a greater accessibility of tetracaine than of procaine to the hydrocarbon region of the membrane and a greater accessibility of procaine than of tetracaine at the membrane's surface. The addition of calcium was found to reverse the quenching of 12-(9-anthroyl)stearic acid by tetracaine in the presence of red cell membranes.     

Study on the interaction between 4-(1H-indol-3-yl)-2-(p-tolyl)quinazoline-3-oxide and human serum albumin

An organic small-molecular drug, 4-(1H-indol-3-yl)-2-(p-tolyl)quinazoline-3-oxide 1a was synthesized. It was employed to investigate the binding interaction and mechanism with human serum albumin (HSA). The experimental results indicated that the fluorescence quenching of HSA by 1a is a static quenching process and formation 1a-HSA complex. The site competition experiments revealed that the combination of 1a on HSA are hydrophobic interactions in the IIA domain and hydrogen bonds in IIIA domain of HSA, and the hydrophobic interactions of 1a on HSA are stronger than that of hydrogen bonds. These results were also confirmed by molecular docking theoretic analysis and ANS-hydrophobic fluorescent probe experiment. Synchronous fluorescence experiments showed that the polarity of HSA microenvironment was increase in the interaction process of 1a with HSA. The results of binding distance explored indicated that the combination distance between 1a and HSA is 3.63 nm, which is between 0.5R₀ and 1.5R₀, revealing the energy transfer between HSA and 1a is non-radiative. These results are very helpful for people to screen out high efficient indoloquinazoline drugs.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE⁻ mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with - presumably - allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

On the mechanism of the PMS-effected quenching of chloroplast fluorescence

Evidence is presented which suggests that N-methylphenazonium methosulfate suppresses the fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts by two mechanisms: (i) indirectly, by catalyzing the buildup of the phosphorylating potential X_E across the thylaknid membrane; (ii) directly, by interacting with excited chlorophyll molecules.Arguments in support of direct quenching are as follows: (i) N-methylphenazonium methosulfate is an efficient quencher of the fluorescence of chlorophyll a in methanol; (ii) the dark-irreversible portion of the light-induced fluorescence lowering in the presence of N-methylphenazonium-methosulfate increases with the concentration of the cofactor, (iii) N-methylphenazonium methosulfate lowers the fluorescence of chloroplasts at an excitation that is too weak to allow formation of X_E.Ascorbate-reduced N-methylphenazonium methosulfate (PMS-SQ) is a more efficient direct quencher of chloroplast fluorescence than oxidized PMS because the thylakoid membrane is more permeable to the reduced species. The permeability to these quenchers is enhanced by the light-induced protonation of the membrane, and suppressed by added Mg²⁺. Different permeability barriers appear to exist for the direct and for the X_E-mediated quenching by N-methylphenazonium methosulfate, since the latter is known to be insensitive to the presence of Mg²⁺.     

ΔpH-Dependent Photosystem II Fluorescence Quenching Induced by Saturating, Multiturnover Pulses in Red Algae

We have previously shown that in the red alga Rhodella violacea, exposure to continuous low intensities of light 2 (green light) or near-saturating intensities of white light induces a ΔpH-dependent PSII fluorescence quenching. In this article we further characterize this fluorescence quenching by using white, saturating, multiturnover pulses. Even though the pulses are necessary to induce the ΔpH and the quenching, the development of the latter occurred in darkness and required several tens of seconds. In darkness or in the light in the presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, the dissipation of the quenching was very slow (more than 15 min) due to a low consumption of the ΔpH, which corresponds to an inactive ATP synthase. In contrast, under far-red illumination or in the presence of 3-(3,4-dichlorophenyl)-1,1′-dimethylurea (only in light), the fluorescence quenching relaxed in a few seconds. The presence of N,N′-dicyclohexyl carbodiimide hindered this relaxation. We propose that the quenching relaxation is related to the consumption of ΔpH by ATP synthase, which remains active under conditions favoring pseudolinear and cyclic electron transfer.     

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.     

Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC–MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal–glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione–alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d₁₁-4-hydroxy-2-(E)-nonenal (d₁₁-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d₁₁-4-HNE-perfused rat livers, the amount of d₁₁-(S)-4-HNE–GSH released from the rat liver was higher than that of d₁₁-(R)-4-HNE–GSH, and more d₁₁-(R)-4-HNE–GSH than d₁₁-(S)-4-HNE–GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.     

Identification of the Chromophores Involved in Aggregation-dependent Energy Quenching of the Monomeric Photosystem II Antenna Protein Lhcb5

Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ.     

Fluorescent ligands derived from 2-(9-anthrylmethylamino)ethyl-appended cyclen for use in metal ion activated molecular receptors

Three new fluorescent ligands derived from 2-(9-anthrylmethylamino)ethyl-appended cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) intended for future use as metal ion activated molecular receptors have been synthesised and characterised. The new ligands, 1,4,7-tris[(2″S)-acetamido-2″-(methyl-3″-phenylpropionate)]-10-(2-N-(9-anthrylmethylamino)ethyl-1,4,7,10-tetraazacyclododecane, 1,4,7-tris[(2″S)-acetamido-2″-(methyl-3″-phenylpropionate)]-10-(2-N-(9-anthrylmethylamino)ethyl-N-[(2″S)-acetamido-2″-(methyl-3″-phenylpropionate])-1,4,7,10-tetraazacyclododecane and 1,4,7-tris[2-hydroxyethyl]-10-(2-N-(9-anthrylmethylamino)ethyl)-N-(2-hydroxyethyl))-1,4,7,10-tetraazacyclododecane, provide the opportunity to investigate the consequences of alkylating the 2-(9-anthrylmethylamino)ethyl fluorophore at the anthrylamine. It was discovered that by doing this the basicity of this amine is lowered and in consequence the pH range over which the PeT induced fluorescence quenching extends is increased by about 1 pH unit. Formation constants were determined in 20% aqueous methanol for the first two ligands with Cd(II) and Cu(II). This demonstrated that alkylation of the anthrylamine significantly increases the stability of the metal complexes.     

Spectroscopic investigation of the interaction of water-soluble azocalix[4]arenes with bovine serum albumin

In this work, three hydrosoluble azocalix[4]arene derivatives, 5-(o-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (o-MAC-Calix), 5-(m-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (m-MAC-Calix) and 5-(p-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (p-MAC-Calix) were synthesized. Their structures were characterized by infrared spectrum (IR), nuclear magnetic resonance spectrum (¹H NMR and ¹³C NMR) and mass spectrum (MS). The interactions between these compounds and bovine serum albumin (BSA) were studied by fluorescence spectroscopy, UV–vis spectrophotometry and circular dichroic spectroscopy. According to experimental results, three azocalix[4]arene derivatives can efficiently bind to BSA molecules and the o-MAC-Calix displays more efficient interactions with BSA molecules than m-MAC-Calix and p-MAC-Calix. Molecular docking showed that the o-MAC-Calix was embedded in the hydrophobic cavity of helical structure of BSA molecular and the tryptophan (Trp) residue of BSA molecular had strong interaction with o-MAC-Calix. The fluorescence quenching of BSA caused by azocalix[4]arene derivatives is attributed to the static quenching process. In addition, the synchronous fluorescence spectroscopy indicates that these azocalix[4]arene derivatives are more accessible to Trp residues of BSA molecules than the tyrosine (Tyr) residues. The circular dichroic spectroscopy further verified the binding of azocalix[4]arene derivatives and BSA.     

(R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST. The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH. The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH.     

Iron specificity of a biosensor based on fluorescent pyoverdin immobilized in sol-gel glass

Two current technologies used in biosensor development are very promising: 1. The sol-gel process of making microporous glass at room temperature, and 2. Using a fluorescent compound that undergoes fluorescence quenching in response to a specific analyte. These technologies have been combined to produce an iron biosensor. To optimize the iron (II or III) specificity of an iron biosensor, pyoverdin (a fluorescent siderophore produced by Pseudomonas spp.) was immobilized in 3 formulations of porous sol-gel glass. The formulations, A, B, and C, varied in the amount of water added, resulting in respective R values (molar ratio of water:silicon) of 5.6, 8.2, and 10.8. Pyoverdin-doped sol-gel pellets were placed in a flow cell in a fluorometer and the fluorescence quenching was measured as pellets were exposed to 0.28 - 0.56 mM iron (II or III). After 10 minutes of exposure to iron, ferrous ion caused a small fluorescence quenching (89 - 97% of the initial fluorescence, over the range of iron tested) while ferric ion caused much greater quenching (65 - 88%). The most specific and linear response was observed for pyoverdin immobilized in sol-gel C. In contrast, a solution of pyoverdin (3.0 μM) exposed to iron (II or III) for 10 minutes showed an increase in fluorescence (101 - 114%) at low ferrous concentrations (0.45 - 2.18 μM) while exposure to all ferric ion concentrations (0.45 - 3.03 μM) caused quenching. In summary, the iron specificity of pyoverdin was improved by immobilizing it in sol-gel glass C.     

Zeaxanthin and the Induction and Relaxation Kinetics of the Dissipation of Excess Excitation Energy in Leaves in 2% O(2), 0% CO(2)

The relationship between the carotenoid zeaxanthin, formed by violaxanthin de-epoxidation, and nonphotochemical fluorescence quenching (q_NP) in the light was investigated in leaves of Glycine max during a transient from dark to light in 2% O₂, 0% CO₂ at 100 to 200 micromoles of photons per square meter per second. (a) Up to a q_NP (which can vary between 0 and 1) of about 0.7, the zeaxanthin content of leaves was linearly correlated with q_NP as well as with the rate constant for radiationless energy dissipation in the antenna chlorophyll (k_D). Beyond this point, at very high degrees of fluorescence quenching, only k_D was directly proportional to the zeaxanthin content. (b) The relationship between zeaxanthin and k_D was quantitatively similar for the rapidly relaxing quenching induced in 2% O₂, 0% CO₂ at 200 micromoles of photons per square meter per second and for the sustained quenching induced by long-term exposure of Nerium oleander to drought in high light (B Demmig, K Winter, A Krüger, F-C Czygan [1988] Plant Physiol 87: 17-24). These findings suggest that the same dissipation process may be induced by very different treatments and that this particular dissipation process can have widely different relaxation kinetics. (c) A rapid induction of strong nonphotochemical fluorescence quenching within about 1 minute was observed exclusively in leaves which already contained a background level of zeaxanthin.     

Specific recognition by the tripeptide lysyl-tryptophyl-α-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558–563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by transPt are required to observe a change in stacking efficiency. In contrast modification by cisPt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cisPt.     

Photoprotective energy quenching in the red alga Porphyridium purpureum occurs at the core antenna of the photosystem II but not at its reaction center

Photosynthetic organisms have evolved light-harvesting antennae over time. In cyanobacteria, external phycobilisomes (PBSs) are the dominant antennae, whereas in green algae and higher plants, PBSs have been replaced by proteins of the Lhc family that are integrated in the membrane. Red algae represent an evolutionary intermediate between these two systems, as they employ both PBSs and membrane LHCR proteins as light-harvesting units. Understanding how red algae cope with light is not only interesting for biotechnological applications, but is also of evolutionary interest. For example, energy-dependent quenching (qE) is an essential photoprotective mechanism widely used by species from cyanobacteria to higher plants to avoid light damage; however, the quenching mechanism in red algae remains largely unexplored. Here, we used both pulse amplitude-modulated (PAM) and time-resolved chlorophyll fluorescence to characterize qE kinetics in the red alga Porphyridium purpureum. PAM traces confirmed that qE in P. purpureum is activated by a decrease in the thylakoid lumen pH, whereas time-resolved fluorescence results further revealed the quenching site and ultrafast quenching kinetics. We found that quenching exclusively takes place in the photosystem II (PSII) complexes and preferentially occurs at PSII’s core antenna rather than at its reaction center, with an overall quenching rate of 17.6 ± 3.0 ns⁻¹. In conclusion, we propose that qE in red algae is not a reaction center type of quenching, and that there might be a membrane-bound protein that resembles PsbS of higher plants or LHCSR of green algae that senses low luminal pH and triggers qE in red algae.     

On the polyphasic quenching kinetics of chlorophyll a fluorescence in algae after light pulses of variable length

This study reports on kinetics of the fluorescence decay in a suspension of the alga Scenedesmus quadricauda after actinic illumination. These are monitored as the variable fluorescence signal in the dark following light pulses of variable intensity and duration. The decay reflects the restoration of chlorophyll fluorescence quenching of the photosystem II (PSII) antennas and shows a polyphasic pattern which suggests the involvement of different processes. The overall quenching curve after a fluorescence-saturating pulse (SP) of 250-ms duration, commonly used in pulse amplitude modulation applications as the tool for estimating the maximal fluorescence (F _m), has been termed P–O, in which P and O have the same meaning as used in the OJIP induction curve in the light. Deconvolution of this signal shows at least three distinguishable exponential phases with reciprocal rate constants of the order of 10, 10², and 10³ ms. The size of the long (>10³ ms) and moderate (~10² ms) lasting components relative to the complete quenching signal after an SP increases with the duration of the actinic pulse concomitantly with an increase in the reciprocal rate constants of the fast (~10 ms) and moderate quenching phases. Fluorescence responses upon single turnover flashes of 30-μs duration (STFs) given at discrete times during the P–O quenching were used as tools for identifying the quencher involved in the P–O quenching phase preceding the STF excitation. Results are difficult to interpret in terms of a single-hit two-state trapping mechanism with distinguishable quenching properties of open and closed reaction centers only. They give support for an earlier hypothesis on a double-hit three-state trapping mechanism in which the so-called semi-closed reaction centers of PSII are considered. In these trapping-competent centers the single reduced acceptor pair [PheQ _A]^1?, depending on the size of photoelectrochemically induced pH effects on the Q _B-binding site, functions as an efficient fluorescence quencher.