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Fluorescence of Photosynthetic Pigments in Vitro
... The longest component lifetime of 5.5 ns could not be reliably determined due to the limited time window. It was, however, similar to the lifetime of monomeric Chl a in solution (Seely and Connolly, 1986), and it is attributed to a small fraction of Chl a molecules whose excited state properties were not affected by the environment. We also observed a similar 5.5 6 3 ns major component under analogous excitation conditions in the pump-probe kinetics of the Chl a dissolved in organic solvents (data not shown). ...
... 1986). This was in good agreement with the measured low fluorescence quantum yield of the Chl a in the b 6 f complex (1.8 6 0.4%, Peterman et al., 1998) that was more than an order of magnitude lower than that of Chl a in methanol (;22%; Seely and Connolly, 1986). Our experiments confirmed that the unusually short Chl* lifetime is also characteristic of the enzymatically active dimeric b 6 f complexes of ML and spinach, as well as inactive monomeric complexes from Synechococcus PCC 7002 suggesting, unexpectedly, that the local environment of the Chl a is similar in both active and inactive forms of the b 6 f complex. ...
... It is well documented that optically excited monomeric Chl a molecules in solution form a long-lived triplet excited state with ;64% yield as a result of intersystem crossing from the singlet excited state (Bowers and Porter, 1967;Seely and Connolly, 1986). Taking into account the measured excited state lifetime of 5-6 ns, the rate of intersystem crossing for Chl a in solvent is ;(9 ns) ÿ1 . ...
... For the CP47 complex the relative number of triplets per pigment is converted to the number of triplets per CP47, by multiplying by 14, the most probable number of pigments in the protein (Barbato et al., 1991; Kwa et al., 1992b). The exact number of photons at a certain laser intensity incident on the sample was calibrated with free Chl a at 77 K for which a triplet quantum yield of 0.64 was assumed (Seely et al., 1986). The ,3-carotene triplet yield was calculated using a triplet extinction coefficient of 2.4 105 M-1cm-' at 530 nm (Bensasson et al., 1983). ...
... The ,3-carotene triplet yield was calculated using a triplet extinction coefficient of 2.4 105 M-1cm-' at 530 nm (Bensasson et al., 1983). The absolute fluorescence quantum yield (DF) was obtained from a comparison of the integral of the emission spectrum from 650 nm to 750 nm with that of free Chl a in Triton X-100 at 77 K, for which a quantum yield of 0.30 was assumed (Seely et al., 1986). In the light of the observed temperature dependence of the Chl fluorescence yield (see Results), the DF of Chl in Triton X-100 at 77 K was compared to DF of Chl in methanol at room temperature. ...
... In the light of the observed temperature dependence of the Chl fluorescence yield (see Results), the DF of Chl in Triton X-100 at 77 K was compared to DF of Chl in methanol at room temperature. With DF (methanol, RT) = 0.22 (Seely et al., 1986), a yield of DF ( ...
Fluorescence emission and triplet-minus-singlet (T-S) absorption difference spectra of the CP47 core antenna complex of photosystem II were measured as a function of temperature and compared to those of chlorophyll a in Triton X-100. Two spectral species were found in the chlorophyll T-S spectra of CP47, which may arise from a difference in ligation of the pigments or from an additional hydrogen bond, similar to what has been found for Chl molecules in a variety of solvents. The T-S spectra show that the lowest lying state in CP47 is at approximately 685 nm and gives rise to fluorescence at 690 nm at 4 K. The fluorescence quantum yield is 0.11 +/- 0.03 at 4 K, the chlorophyll triplet yield is 0.16 +/- 0.03. Carotenoid triplets are formed efficiently at 4 K through triplet transfer from chlorophyll with a yield of 0.15 +/- 0.02. The major decay channel of the lowest excited state in CP47 is internal conversion, with a quantum yield of about 0.58. Increase of the temperature results in a broadening and blue shift of the spectra due to the equilibration of the excitation over the antenna pigments. Upon increasing the temperature, a decrease of the fluorescence and triplet yields is observed to, at 270 K, a value of about 55% of the low temperature value. This decrease is significantly larger than of chlorophyll a in Triton X-100. Although the coupling to low-frequency phonon or vibration modes of the pigments is probably intermediate in CP47, the temperature dependence of the triplet and fluorescence quantum yield can be modeled using the energy gap law in the strong coupling limit of Englman and Jortner (1970. J. Mol. Phys. 18:145-164) for non-radiative decays. This yields for CP47 an average frequency of the promoting/accepting modes of 350 cm-1 with an activation energy of 650 cm-1 for internal conversion and activationless intersystem crossing to the triplet state through a promoting mode with a frequency of 180 cm-1. For chlorophyll a in Triton X-100 the average frequency of the promoting modes for non-radiative decay is very similar, but the activation energy (300 cm-1) is significantly smaller.
... Its amplitude is 3.5% compared with the contribution at 684 nm. Given that the fluorescence yield of monomeric Chl a is 0.30 (Seely, et al., 1986) and that of the main emission of the RC at 684 nm is 0.07 (see below), it probably arises from about 1% of the pigments. This very small contribution is neglected in this paper.Fig. ...
... The decay of the excited state of Chl is determined by the sum of rate constants for radiative decay (kR), intersystem crossing to the metastable triplet state (k1sc), and internal conversion into heat (k1c). For free Chl a, kR can be calculated with the Strickler-Berg relation or from the quantum yield of fluorescence (0.30) and the excited state lifetime (6 ns) (see Seely et al., 1986), i.e., kR-0.05 ns-. ...
... ns-. The ratio of kIsc to kR is about 2:1 (Seely et al., 1986), i.e., k1sc-0 From the temperature dependence of the fluorescence yield it is evident that at T -40 K C681 becomes thermally connected to the other pigments. Because P680 is included in this set of other pigments, the fluorescence yield decreases. ...
A key step in the photosynthetic reactions in photosystem II of green plants is the transfer of an electron from the singlet-excited chlorophyll molecule called P680 to a nearby pheophytin molecule. The free energy difference of this primary charge separation reaction is determined in isolated photosystem II reaction center complexes as a function of temperature by measuring the absolute quantum yield of P680 triplet formation and the time-integrated fluorescence emission yield. The total triplet yield is found to be 0.83 +/- 0.05 at 4 K, and it decreases upon raising the temperature to 0.30 at 200 K. It is suggested that the observed triplet states predominantly arise from P680 but to a minor extent also from antenna chlorophyll present in the photosystem II reaction center. No carotenoid triplet states could be detected, demonstrating that the contamination of the preparation with CP47 complexes is less than 1/100 reaction centers. The fluorescence yield is 0.07 +/- 0.02 at 10 K, and it decreases upon raising the temperature to reach a value of 0.05-0.06 at 60-70 K, increases upon raising the temperature to 0.07 at approximately 165 K and decreases again upon further raising the temperature. The complex dependence of fluorescence quantum yield on temperature is explained by assuming the presence of one or more pigments in the photosystem II reaction center that are energetically degenerate with the primary electron donor P680 and below 60-70 K trap part of the excitation energy, and by temperature-dependent excited state decay above 165 K. A four-compartment model is presented that describes the observed triplet and fluorescence quantum yields at all temperatures and includes pigments that are degenerate with P680, temperature-dependent excited state decay and activated upward energy transfer rates. The eigenvalues of the model are in accordance with the lifetimes observed in fluorescence and absorption difference measurements by several workers. The model suggests that the free energy difference between singlet-excited P680 and the radical pair state P680+l- is temperature independent, and that a distribution of free energy differences represented by at least three values of about 20, 40, and 80 meV, is needed to get an appropriate fit of the data.
... For the vibronically excited spectra, the power could be increased to 2 mW/cm 2 . Fluorescence quantum yields were determined from the ratio of the areas of emission of Cytb 6 f and Chl a (Sigma) in methanol (fluorescence yield 22%; Seely and Connolly, 1986). The areas were corrected for the amount of absorbed photons at the excitation wavelength. ...
... e from 655 to 684 nm, the anisotropy is FIGURE 1 Absorption spectrum of air-oxidized Cytb 6 f at 4 K. The spectral bandwidth was 1 nm. The inset shows the Chl Q y -region of the spectrum as a function of temperature. The highest, narrowest spectrum is at 4 K, the lowest and broadest is at 275 K. In between are 50, 100, 175, and 225 K, respectively.Seely and Connolly, 1986), the former can be calculated to be 1.8 0.4%. This quantum yield is independent of the redox states of the hemes, which were modified by the consecutive addition of ferricyanide, ascorbate, and dithionite. Interestingly, the fluorescence quantum yield increased to the same value as for Chl in methanol after the addition of Triton X-100 ...
... The whole data set could be fitted satisfactorily with one decay time: 250 20 ps. This fluorescence lifetime is about a factor of 20 less than that of Chl a in a " normal " protein environment or methanol (5– 6 ns; Seely and Connolly, 1986), indicating that the Chl is specifically bound. No nanosecond component was needed to fit the data, which indicates that the amount of Chl that is specifically bound to the complex is larger than 95%. ...
A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1.
... The longest component lifetime of 5.5 ns could not be reliably determined due to the limited time window. It was, however, similar to the lifetime of monomeric Chl a in solution (Seely and Connolly, 1986), and it is attributed to a small fraction of Chl a molecules whose excited state properties were not affected by the environment. We also observed a similar 5.5 6 3 ns major component under analogous excitation conditions in the pump-probe kinetics of the Chl a dissolved in organic solvents (data not shown). ...
... 1986). This was in good agreement with the measured low fluorescence quantum yield of the Chl a in the b 6 f complex (1.8 6 0.4%, Peterman et al., 1998) that was more than an order of magnitude lower than that of Chl a in methanol (;22%; Seely and Connolly, 1986). Our experiments confirmed that the unusually short Chl* lifetime is also characteristic of the enzymatically active dimeric b 6 f complexes of ML and spinach, as well as inactive monomeric complexes from Synechococcus PCC 7002 suggesting, unexpectedly, that the local environment of the Chl a is similar in both active and inactive forms of the b 6 f complex. ...
... It is well documented that optically excited monomeric Chl a molecules in solution form a long-lived triplet excited state with ;64% yield as a result of intersystem crossing from the singlet excited state (Bowers and Porter, 1967;Seely and Connolly, 1986). Taking into account the measured excited state lifetime of 5-6 ns, the rate of intersystem crossing for Chl a in solvent is ;(9 ns) ÿ1 . ...
The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.
... In Appendix 2 this factor (C) is determined. The radiative lifetime is estimated from literature values for the fluorescence quantum yield and the fluorescence lifetime of Chl a (Seely and Conolly, 1986), leading to C ϭ 42/n 4 ps Ϫ1 nm 6 for r D ϭ 18.5 ns (for details see Appendix 2). The refractive index can be used for the actual scaling of the calculated time constants to "real" time constants. ...
... The radiative rate in the Förster equation is estimated by using k r D ϭ f / f , where f is the fluorescence lifetime and f is the quantum yield for fluorescence. Using literature values for f and f for Chl a in methanol and in ether, we find an average radiative lifetime of 18.5 ns (Seely and Conolly, 1986). So for C we find a value of 42/n 4 ps Ϫ1 nm 6 . ...
Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Förster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Förster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.
... Similarly, the fluorescence spectrum of the same chlorophyll a solution was obtained at room temperature in the range 400-800 nm, at an excitation wavelength exc = 370 nm. A broad band was observed with a peak maximum at 667 nm, which coincided with the characteristic red fluorescence band of chlorophyll a that has been reported in the literature [30,44]. ...
Both chlorophyll and the heme group of hemoglobin possess a central tetrapyrrolic structure formed by four pyrroles bonded to methine groups. It is important to stress that natural and synthetic tetrapyrroles involve interesting properties; and sometimes, in order to deploy them, it is convenient to trap these macrocycles within pore networks. Due to their nature, these molecules cannot be inserted by diffusion inside pores and the classical impregnation method can just render lowly concentrated and heterogeneous materials. To overcome this difficulty our research group has been working in the development of sol–gel methodologies for effectively inserting synthetic tetrapyrrolic species inside inorganic pore networks. In this way, it has been found that the average pore size and specific surface area of the trapping substrate depend on the cation identity and structure of the macrocyclic complex. Yet, the interactions of the captured species with the M-OH surface groups resting at the pore walls affect the efficient display of the properties of these species. The physicochemical properties of the captured macrocycle remain similar to those observed in solution by situating the species far from the M-OH groups or substituting these by alkyl or aryl groups. Here, we present the first results of the application of the above methodology for the covalent bonding of chlorophyll a in stable and monomeric form inside translucent organo modified silica xerogels and mesoporous SBA-15 substrates. In these systems, chlorophyll remains fixed to the silica pore surface while its UV absorption and fluorescence spectra remain similar to those displayed in solution. The optimization of the synthesis of materials based on the trapping of natural chlorophyll could be a crucial step in the development of novel photoreactive, optical, catalytic, sensoring, and medical devices.
... In leaves, clear and distinct effects of O 2 pressures (but not N 2 pressures) are seen on the different levels of fluorescence achieved during prolonged induction in the light (Vidaver et al. 1981a,b). In vitro, in some solvents, the rate constant of oxygen interaction with the excited state of Chl a approaches a value of 10 10 M 1 s 1 (Seely and Connolly 1986). Oxygen is much more soluble in hydrophobic phases than in water (Hildebrand and Scott 1964) and, if restricted to the membrane volume, the concentration of oxygen released after the third flash (1 O 2 per RC) would be 0.5 mM. ...
Photosystem II (PS II) of plants and cyanobacteria, which catalyzes the light-induced splitting of water and the release of oxygen, is the primary source of oxygen in the earth atmosphere. When activated by short light flashes, oxygen release in PS II occurs periodically with maxima after the third and the seventh flashes. Many other processes, including chlorophyll (Chl) t a fluorescence, are also modulated with period of four, reflecting their sensitivity to the activity of Photosystem II. A new approach has been developed for the analysis of the flash-induced fluorescence of Chl t a in plants, which is based on the use of the generalized Stern–Volmer equation for multiple quenchers. When applied to spinach thylakoids, this analysis reveals the presence of a new quencher of fluorescence whose amplitude is characterized by a periodicity of four with maxima after the third and the seventh flashes, in phase with oxygen release. The quencher appears with a delay of 0.5 ms followed by a rise time of 1.2–2 ms at pH 7, also in agreement with the expected time for oxygen evolution. It is concluded that the quencher is a product of the reaction leading to the oxygen evolution in PS II. The same quenching activity, maximal after the third flash, could be seen in dark adapted leaves, and provides the first fully time-resolved measurement of the kinetics of the oxygen evolution step in the leaf. Thus, the non-invasive probe of Chl t a fluorescence provides a new and sensitive method for measuring the kinetics of oxygen evolution with potential for use in plants and cyanobacteria t in vivo.
... The lack of 1 O 2 * luminescence in the b-DM preparation from B. corticulans may be best explained by the significantly low quantum yield of 3 Chl a*, which is in turn accounted for by the drastically reduction in the Chl a fluorescence lifetime, i.e. from *5 ns for Chl a free in organic solvents (Seely and Connolly 1986) to *240 ps for it bound in the Cyt b 6 f complex . In fact, the 1 Chl a* lifetime had been reported to be *250 ps for the Cyt b 6 f complex from Synechocystis PCC6803 (Peterman et al. 1998) and *190 ps for the highly enzymatically active Cyt b 6 f complex from M. laminosus . ...
We have attempted to investigate the correlation between the detergent-perturbed structural integrity of the Cyt b
6f complex from the marine green alga Bryopsis corticulans and its photo-protective properties, for which the nonionic detergents n-octyl-β-d-glucopyranoside (β-OG) and n-dodecyl-β-d-maltoside (β-DM), respectively, were used for the preparation of Cyt b
6f, and the singlet oxygen (1O2*) production as well as the triplet excited-state chlorophyll a (3Chl a*) formation and deactivation were examined by spectroscopic means. Near-infrared luminescence of 1O2* (~1,270 nm) on photo-irradiation was detected for the β-OG preparation where the complex is mainly in oligomeric state, but not for the β-DM one in which the complex exists in dimeric form. Under anaerobic condition, photo-excitation of Chl a in the β-DM preparation generated 3Chl a* with a lower quantum yield of ΦT ~ 0.02 and a longer lifetime of ~600 μs with respect to those as in the case of β-OG preparation, ΦT ~ 0.12 and 200–300 μs. These results prove that the enzymatically active and intact Cyt b
6f complex on photo-excitation tends to produce little 3Chl a* or 1O2*, which implies that the pigment–protein assembly of Cyt b
6f complex per se is crucial for photo-protection.
... However, it can not be excluded that the chl a aggregates or chlorophylls unspecifically adsorbed to the proteins might contribute to the fluorescence at 730 nm. Such low temperature fluorescence emission at 725±730 nm was reported for chl a aggregates in Triton X-100 micelles or chl a molecules associated with nonphotosynthetic proteins [35]. Also, the part of the emission maximum at 660 nm might derive from free chl a. ...
The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.
... The same is seen with the comparison of fluorescence halfwidth in pure solvents ( Table 4). The fluorescence lifetime is also solvent dependent (41), being lowest in alcohols and highest in aprotic polar solvents. In the case of nondegassed polar solvents, practically no difference exists between L 1 and L 1(2) H solvates. ...
The conversion of chlorophyll a (Chl a) monomers into large aggregates in six polar solvents upon addition of water has been studied by means of absorption, fluorescence spectroscopy and fluorescence lifetime measurements for the purpose of elucidating the various environmental factors promoting Chl a self-assembly and determining the type of its organization. Two empirical solvent parameter scales were used for quantitative characterization of the different solvation properties of the solvents and their mixtures with water. The mole fractions of water f1/2 giving rise to the midpoint values of the relative fluorescence quantum yield were determined for each solvent, and then various solvent-water mixture parameters for the f1/2 values were compared. On the basis of their comparison, it is concluded that the hydrogen-bonding ability and the dipole-dipole interactions (function of the dielectric constant) of the solvent-water mixtures are those that promote Chl a self-assembly. The influence of the different nature of the non-aqueous solvents on the Chl aggregation is manifested by both the different water contents required to induce Chl monomer-->aggregate transition and the formation of two types of aggregates at the completion of the transition: species absorbing at 740-760 nm (in methanol, ethanol, acetonitrile, acetone) and at 667-670 nm (in pyridine and tetrahydrofuran). It is concluded that the type of Chl organization depends on the coordination ability and the polarizability (function of the index of refraction) of the organic solvent. The ordering of the solvents with respect to the f1/2 values--methanol < ethanol < acetonitrile < acetone < pyridine < tetrahydrofuran--yielded a typical lyotropic (Hofmeister) series. On the basis of this solvent ordering and the disparate effects of the two groups of solvents on the Chl a aggregate organization, it is pointed out that the mechanism of Chl a self-assembly in aqueous media can be considered a manifestation of the Hofmeister effect, as displayed in the lipid-phase behavior (Koynova et al., Eur. J. Biophys. 25, 261-274, 1997). It relates to the solvent ability to modify the bulk structure and to distribute unevenly between the Chl-water interface and bulk liquid.
... The effects of solvent on uorescence emission peak shifts have been shown (26). In general, pure Chl a molecules (in solvent) emit uorescence in the red and far-red regions, with emission maxima shifted toward wavelengths shorter than those for the emission maxima of intact plants. ...
Fecal contamination of food products is a critical health issue. To test the feasibility of the use fluorescent techniques to detect fecal contamination, fluorescence excitation and emission characteristics of fecal matter from cows, deer, swine, chickens, and turkeys in the UV to far-red regions of the spectrum were evaluated. To allow the optimization of the detection of fecal contamination on animal carcasses and cut meats, emission-excitation spectra of the feces were compared with spectra for animal meats. The feedstuffs for the swine, chickens, and turkeys were also analyzed. Excitation at approximately 410 to 420 nm yielded the highest level of fluorescence for both feces and feedstuffs. Emission maxima were in the red region (at 632 nm for chicken feces and at 675 nm for the feces of the other species). The major constituent responsible for emission at 632 nm was tentatively identified as protoporphyrin IX; emission at 675 nm most likely emanates from chlorophyll a or its metabolites. Animal meats emitted strong fluorescence in the blue-green regions, but no emission peaks were observed in the red region for these meats. These results suggest that fluorescence emissions from naturally occurring chlorophyll a and its metabolites are good markers for fecal contamination and that with excitation at 410 to 420 nm, the responses of fecal matter can easily be differentiated from the responses of animal meats. We suggest that the detection of fecal contamination can be enhanced by requiring a minimum chlorophyll a content in the finishing diets of all farm animals.
... It was shown that the Chl a molecule in the cytochrome b 6 f complex in solution has an unusually short singlet excited state lifetime of ~200 ps that is characteristic of both the enzymatically active dimeric b 6 f complexes 14 and the functionally inactive monomeric cytochrome b 6 f complexes. 12,14 The 200 ps excited state lifetime contrasts with the 5 -6 ns lifetime reported for monomeric Chl a in solution, 28 which is ~25-fold longer. After a thorough examination of possible causes of the observed rapid quenching of the Chl a singlet excited state, it was inferred that the excitation-induced electron transfer between the Chl a and nearby aromatic amino acid residue(s) is the most likely explanation for the observed effect. ...
The cytochrome b
6f complex of oxygenic photosynthesis contains a single chlorophyll a molecule. The singlet excited state of the Chl a. molecule is quenched by the surrounding protein matrix, and thus the lifetime of this state may serve as a probe of the proteins structure. In this work, singlet excited state dynamics were measured in well-diffracting crystals using femtosecond time-resolved optical pump-probe methodology. Lifetimes of the Chl a molecule in crystals of the cytochrome b
6f complex having different space groups were 3–6 times longer than those determined in detergent solution of the b
6f. The observed differences in excited state dynamics may arise from small (1–1.5 Å) changes in local protein structure caused by crystal packing. The Chl a excited state lifetimes measured in dissolved cytochrome b
6f complexes from several different species are essentially the same, in spite of differences in the local amino acid sequences around the Chl a. This supports an earlier hypothesis that the short excited state lifetime of Chl a is critical for the function of the b
6f complex.
The design of new molecules for photochemical studies typically requires knowledge of spectral features of pertinent chromophores beginning with the absorption spectrum (λabs) and accompanying molar absorption coefficient (ε, M⁻¹cm⁻¹) and often extending to the fluorescence spectrum (λem) and fluorescence quantum yield (Φf), where the fluorescence properties may be of direct relevance or useful as proxies to gain insight into the nature of the first excited singlet state. PhotochemCAD databases, developed over a period of 30 years, are described here. The previous databases for 150 compounds have been expanded to encompass 339 compounds for which absorption spectra (including ε values), fluorescence spectra (including Φf values) and references to the primary literature have been included where available (551 spectra altogether). The compounds exhibit spectra in the ultraviolet, visible, and/or near-infrared spectral regions. The compound classes and number of members include acridines (21), aromatic hydrocarbons (41), arylmethane dyes (11), azo dyes (18), biomolecules (18), chlorins/bacteriochlorins (16), coumarins (14), cyanine dyes (19), dipyrrins (7), heterocycles (26), miscellaneous dyes (13), oligophenylenes (13), oligopyrroles (6), perylenes (5), phthalocyanines (11), polycyclic aromatic hydrocarbons (16), polyenes/polyynes (10), porphyrins (34), quinones (24), and xanthenes (15). A database of 31 solar spectra also is included.
Leaves under stressful conditions usually show downregulated maximum quantum efficiency of photosystem II [inferred from variable to maximum chlorophyll (Chl) a fluorescence (Fv/Fm), usually lower than 0.8], indicating photoinhibition. The usual method to evaluate the degree of photoinhibition in winter red leaves is generally by measuring the Fv/Fm on the red adaxial surface. Two phenotypes of overwintering Buxus microphylla ‘Wintergreen’ red leaves, with different measuring site and leaf thickness, were investigated in order to elucidate how red pigments in the outer leaf layer affected the Chl a fluorescence (Fv/Fm) and photochemical reflectance index. Our results showed that the Fv/Fm measured on leaves with the same red surface, but different leaf thickness, exhibited a slightly lower value in half leaf (separated upper and lower layers of leaves by removing the leaf edge similarly as affected by winter freezing and thawing) than that in the intact leaf (without removing the leaf edge), and the Fv/Fm measured on the red surface was significantly lower than that on the inner or backlighted green surface of the same thickness. Our results suggest that the usual measurement of Fv/Fm on red adaxial surface overestimates the actual degree of photoinhibition compared with that of the whole leaf in the winter.
This chapter presents a review of primary charge separation processes in various photosynthetic reaction centers. Common motif of the known reaction centers is briefly discussed, followed by a comprehensive overview of the charge separation mechanisms in three major reaction center complexes for which crystal structures have been determined: bacterial reaction center, photosystem II, and photosystem I.
Photosynthesis converts solar energy into energy of chemical bonds. This process is initiated when a photon of sunlight is absorbed by a photosynthetic pigment molecule, followed by a highly efficient transfer of the excitation energy, excitation trapping, and charge separation at the reaction center. The excited state dynamics initiated by light absorption are central to the primary reactions of photosynthesis. Unless successfully transferred away from the excited chromophore within the excitation lifetime, the excitation energy relaxes back to the electronic ground state, either via emission of a photon (radiative decay) or through various nonradiative processes. The photosynthetic machinery can control the nonradiative relaxation rate: it can increase it under stress conditions (e.g., high light) by adjusting electronic properties of chromophores as well as their interaction, or decrease it under optimal conditions reaching >90 % efficiency of energy transfer. Some background in photophysics is, therefore, needed to understand the mechanistic aspects of the initial events following photoexcitation of photosynthetic complexes. The goal of this chapter is to describe the excited states involved in photoreactions and to outline the physical basis of photophysical processes involved in photosynthesis. We introduce the principles of light absorption and the nature of electronic excited states and light-initiated dynamics in photosynthetic complexes. De-excitation pathways, rate constants, quantum yields and lifetimes of fluorescence, excitation energy transfer and related photophysics are discussed. In the concluding section, we present an overview of the mechanisms of non-photochemical quenching (NPQ) of chlorophyll fluorescence in terms of photophysics of the excited states of photosynthetic pigments.
Photosynthesis, the conversion of light energy into stabilized chemical energy, involves the absorption of light by a pigment, energy transfer, energy trapping or stabilization by reaction centers, and the initiation of chemical reactions from donor to acceptor molecules. The process continues with a sequence of oxidation-reduction reactions that compose the electron transport that leads to the formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in plants or reduced NAD (NADH) in bacteria and adenosine triphosphate (ATP). Reactions leading to the fixation of carbon are powered by the energy available in the molecules of NADPH (or NADH) and ATP.
Chemical structures and distribution of chlorophylls and bacteriochlorophyllsPheophytins and bacteriopheophytinsChlorophyll biosynthesisSpectroscopic properties of chlorophyllsCarotenoidsBilins
In the first part of this work, a brief review is presented of the recent knowledge of charge stabilization processes in flash-excited reaction center protein from photosynthetic bacteria Rhodobacter sphaeroides. The adaptation of the protein to charge separation is comprised of different manifestations of protein relaxation including proton binding at the late phase. In the second part, a unique method, comparative measurement of prompt and millisecond-delayed fluorescence of the bacteriochlorophyll dimer of the reaction center protein is used to determine the free energy levels of the charge-separated states with respect to that of the excited singlet state of the dimer: −910 ± 20 meV and −970 ± 20 meV were measured at pH 8.0 for reaction centers in the absence and presence of secondary quinone, respectively. The pH-dependence of the energetics of charge stabilization due to light-induced proton binding is described in reaction centers with and without secondary quinone activity. The range of free-energy change between pH 11 and pH 5 was −65 meV with a single (alkaline) inflection point (no active secondary quinone) and −175 meV with an additional (acidic) inflection point (active secondary quinone). The conclusions from fluorescence data agreed well with pH-dependence of integrated proton uptake (a model-independent method) and with calculations based on interaction of quinones with four key amino acid residues. The enthalpy and entropy parts of the free-energy changes were determined from van't Hoff analysis of the delayed fluorescence and compared with data of other methods. It is concluded that the charge stabilization including proton binding is a highly enthalpy-driven process.
(Bacterio)Chlorophyll ((B)Chl) molecules play a major role in photosynthetic light harvesting proteins and the knowledge of their triplet state energies is essential to understand the mechanisms of photodamage and photoprotection, as the triplet excitation energy of (B)Chl molecules can readily generate highly reactive singlet oxygen. The triplet state energies of ten natural chlorophyll (Chl a, b, c2, d) and bacteriochlorophyll (BChl a, b, c, d, e, g) molecules and one bacteriopheophytin (BPheo g) have been directly determined via their phosphorescence spectra. Phosphorescence of four molecules (Chl c2, BChl e and g, BPheo g) was characterized for the first time. Additionally, the relative phosphorescence to fluorescence quantum yield for each molecule was determined. The measurements were performed at 77K using solvents providing a six-coordinate environment of the Mg2+ ion, which allows direct comparison of these (B)Chls. Density functional calculations of the triplet state energies show good correlation with the experimentally determined energies. The correlation determined computationally was used to predict the triplet energies of three additional (B)Chl molecules: Chl c1, Chl f, and BChl f.
Absorption and fluorescence spectra were measured for Chlamydobotrys stellata cultured either photo-heterotrophically on acetate or autotrophically on CO2 as well as during adaptation from hetero- to autotrophic conditions. Curve analyses of the absorption spectra at liquid nitrogen temperature suggest the presence of chlorophyll-a forms with their main absorption peaks at 663, 670, 678, 685, 693 and 707 nm. The proportion of the longer wavelength forms, 685, 693 and 707 nm, decreases during adaptation to autotrophic growth. The chlorophyll-b content of the photo-heterotrophic culture was very low.
The freshwater blooms mainly blue-green algal blooms occur frequently in the lower Naktong River in summer, which provoke many socio-economical problems; therefore, the early detection of bloom events are demanding through the quantitative and qualitative analyses of blue green algal species. The in vivo fluorescence properties of cultured strains of Microcystis aeruginosa, M. viridis, M. wesenbergii, M. ichthyoblabe, Anabaena cylindrica, A. flos-aquae, and Synedra sp. were investigated. Wild phytoplankton communities of the lower Naktong River were also monitored at four stations in terms of their standing stocks, biomass and fluorescence properties compared with its absorption spectram. The 77K fluorescence emission spectra of each cultured strains normalized at 620 nm was very specific and enabled to detect of blue green algal biomass qualitatively and quantitatively. The relative chlorophyll a concentration determined by chlorophyll fluorescence analysis method showed significant relationship with chlorophyll a concentration determined by solvent extraction method ( = 0.906), and the blue-green algal cell number determined by microscopic observation ( = 0.588), which gives insight into applications to early detection of blue green algal bloom.
Solutions of free base meso-tetraphenylporphyrin (H2TPP), meso-tetra(4-carboxyphenyl)porphyrin (H2TPPC), and meso-tetra(4-pyridyl)porphyrin (H2TPyP) under various conditions generate aggregates whose absorption spectra are characterized by invariant Soret bands with bandwidths that are independent of the preparative method. One of the Soret bands is blue-shifted (H-aggregate) relative to the monomeric porphyrin band; other Soret bands are red-shifted (J-aggregates). The aggregates are characterized by different nonradiative rate constants for excited singlet-state decay and by different efficiencies of singlet−singlet annihilation at the high energies of laser excitation. The quantum yields of fluorescence vary between 10-5 and 10-2, and the corresponding fluorescence lifetimes vary in the range from 10-12 to 10-9 s; they are more than 1 order of magnitude smaller than those of the corresponding monomeric porphyrins. Lifetimes (τ) correlate with the characteristic ground-state absorption recovery times of the aggregates. The sizes of the H-aggregate and aggregates that are characterized by a minimal blue shift of the Soret band range from 15 to 27 Å.
Only recently have various methodological approaches been successful in producing Raman spectra of chlorophylls resonantly with their lowest singlet electronic transitions (Qy bands). The information content of these new approaches and their impact on current research on photosynthetic proteins are discussed. It is shown that resonance methods can contribute very valuable data on the dynamics of the primary photophysical events, while preresonance methods are yielding many new data on the structural bases of these events at atomic level. © 1995 John Wiley & Sons, Inc.
The fluorescence anisotropy of photosystem I particles, isolated from spinach chloroplasts and containing approximately 200 chlorophyll molecules per reaction center, has been investigated at low temperatures. Fluorescence anisotropy has been measured upon excitation with laser lines at 476.5 and 632.8 nm. Using our data for the fluorescence anisotropy at these conditions and the new `practical' formula for the degree of polarization of a triple-chromophore complex under steady-state excitation, derived recently by Demidov, we estimate the mutual orientation of absorbing chromophores and long wavelength pigments—chlorophyll a molecules, that absorb at wavelengths longer than the corresponding reaction center, in Photosystem I particles. The angle between the transition dipole moments of chlorophyll a, belonging to the light-harvesting complex of PS I and absorbing the excitation at 632.8 nm, and the emitting long wavelength pigment at 735 nm is estimated to be 40°, whereas the angle between the transition dipole moments of chlorophyll b, belonging to the light-harvesting complex of PS I and absorbing the excitation at 476.5 nm, and the emitting long wavelength pigment at 735 nm—60°.
Abstract— Polarized absorption, fluorescence and photoacoustic spectra of bacteriochlorophyll (BChl)-lipoprotein complexes from the purple bacterium Chromatium minutissimum oriented in stretched polyvinylalcohol films were measured at room temperature and 85 K. The preparations contain large amounts of the B800-820 antenna complexes. From polarized absorption spectra taken under various light beam incidence angles with respect to the film plane, conclusions concerning arrangement of pigment molecules in B800-820 complex are obtained. The transition moments of the BChl Qy band are not exactly parallel to the membrane plane. It seems that there are pools of differently oriented BChl chromophores absorbing in both 800 nm and 820 nm regions. Change in temperature strongly influences linear dichroism of carotenoids and BChl Qy bands. The reversible changes in absorption, linear dichroism and photoacoustic spectra caused by the variation in sample temperature suggest strongly the reversible twisting of carotenoid molecules, related probably to modification of the interactions between carotenoids and proteins. Various carotenoids exhibit different yield of thermal deactivation and this yield is also temperature dependent.
The conversion of chlorophyll a (Chl a) monomers into large aggregates in six polar solvents upon addition of water has been studied by means of absorption, fluorescence spectroscopy and fluorescence lifetime measurements for the purpose of elucidating the various environmental factors promoting Chl a self-assembly and determining the type of its organization. Two empirical solvent parameter scales were used for quantitative characterization of the different solvation properties of the solvents and their mixtures with water. The mole fractions of water f1/2 giving rise to the midpoint values of the relative fluorescence quantum yield were determined for each solvent, and then various solvent–water mixture parameters for the f1/2 values were compared. On the basis of their comparison, it is concluded that the hydrogen-bonding ability and the dipole–dipole interactions (function of the dielectric constant) of the solvent–water mixtures are those that promote Chl a self-assembly. The influence of the different nature of the nonaqueous solvents on the Chl aggregation is manifested by both the different water contents required to induce Chl monomer → aggregate transition and the formation of two types of aggregates at the completion of the transition: species absorbing at 740–760 nm (in methanol, ethanol, acetonitrile, acetone) and at 667–670 nm (in pyridine and tetrahydrofuran). It is concluded that the type of Chl organization depends on the coordination ability and the polarizability (function of the index of refraction) of the organic solvent. The ordering of the solvents with respect to the f1/2 values—methanol < ethanol < acetonitrile < acetone < pyridine < tetrahydrofuran—yielded a typical lyotropic (Hofmeister) series. On the basis of this solvent ordering and the disparate effects of the two groups of solvents on the Chl a aggregate organization, it is pointed out that the mechanism of Chl a self-assembly in aqueous media can be considered a manifestation of the Hofmeister effect, as displayed in the lipid-phase behavior (Koynova et al., Eur. J. Biophys. 25, 261–274, 1997). It relates to the solvent ability to modify the bulk structure and to distribute unevenly between the Chl–water interface and bulk liquid.
The light driven electron transfer reactions of photosynthesis are best characterized in the reaction centers of purple nonsulfur
bacteria such as Rhodobacter sphaeroides. These reactions are extraordinarily fast: the first of them taking place within a few picoseconds. Because photosynthetic
electron transfer is initiated by light, and the redox active cofactors have distinct spectral signatures, both the kinetics
and thermodynamics of the reaction can be studied on the timescale of the actual reactions. This, in combination with the
availability of detailed structural information, has made the electron transfer reactions of purple nonsulfur bacteria some
of the most completely characterized electron transfer reactions in biological systems. In addition to the spectroscopic tools
which have been used to characterize the electron transfer reactions, the reaction center has also been the subject of mutagenesis
studies. The ease with which genetic engineering can be performed in these bacteria, coupled with the structurally robust
nature of the reaction center, has resulted in many mutations which affect the early electron transfer reactions. Critical
protein-cofactor interactions have now been identified and analyzed which affect the metal content, redox potentials, electronic
structure and intermolecular coupling of the bacteriochlorophylls and bacteriopheophytins of the reaction center. In combination,
these studies have suggested various possible mechanisms for early electron transfer which involve both the electronic states
of the cofactors as well as nuclear conformational changes in the surrounding protein.
Photoinhibition under aerobic and anaerobic conditions was analyzed in O2-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680+ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced QA(QA
-). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated QA (QAH2). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680*QA P680+QA
-), is nearly the same in O2-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the QAH2-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of QA leading to QAH2 formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.
The spectroscopy characteristics and the fluorescence lifetime for the chloroplasts isolated from the pseudo ginseng, water
hyacinth and spinach plant leaves have been studied by absorption spectra, low temperature steady-state fluorescence spectroscopy
and single photon counting measurement under the same conditions and by the same methods. The similarity of the absorption
spectra for the chloroplasts at room temperature suggests that different plants can efficiently absorb light of the same wavelength.
The fluorescence decays in PS II measured at the natural QA state for the chloroplasts have been fitted by a three-exponential kinetic model. The three fluorescence lifetimes are 30,
274 and 805 ps for the pseudo ginseng chloroplast; 138, 521 and 1494 ps for the water hyacinth chloroplast; 197, 465 and 1459
ps for the spinach chloroplast, respectively. The slow lifetime fluorescence component is assigned to a collection of associated
light harvesting Chl a/b proteins, the fast lifetime component to the reaction center of PS II and the middle lifetime component
to the delay fluorescence of recombination of P+
680 and Pheo-. The excitation energy conversion efficiency(η) in PS II RC is defined and calculated on the basis of the 20 ps electron transfer time constant model, 60%, 87% and 91%
for the pseudo ginseng, water hyacinth and spinach chloroplasts, respectively. This interesting result is in unconformity
with what is assumed to be 100% efficiency in PS II RC. Our result in this work stands in line with the 20 ps electron transfer
time constant in PS II rather sound and the water hyacinth plant grows slower than the spinach plant does as envisaged on
the efficiency. But, our results predict that those plants can perform highly efficient transfer of photo-excitation energy
from the light-harvesting pigment system to the reaction center (closely to 100%). The conclusion contained in this paper
reveals the plant growth characteristics expressed in the primary processes of photosynthesis and a relationship between a
plant growing rate and its spectroscopy characteristics and fluorescence lifetimes, namely, the slower a plant grows, the
less excitation energy conversation efficiency used might be anticipated.
We present a simple approach for the calculation of in vivo fluorescence excitation spectra from measured absorbance spectra of the isolated pigments involved. Taking into account shading of the pigments by each other, energy transfer from carotene to chlorophyll a, and light scattering by the leaf tissue, we arrive at a model function with 6 free parameters. Fitting them to the measured fluorescence excitation spectrum yields good correspondence between theory and experiment, and parameter estimates which agree with independent measurements. The results are discussed with respect to the origin and the interpretation of in vivo excitation spectra in general.
Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.
— Absorption and emission spectra of bacteriochlorophyll c dissolved in a variety of solvents were measured and fluorescence quantum yields determined from the integrated emission spectra. Values for the emission maxima calculated from the positions and bandwidths of the red absorption band using the Stepanov relationship agreed closely with the experimental values. Fluorescence quantum yields varied between 0.10 in methanol and 0.36 in tetrahydrofuran and in dibutylamine. Fluorescence lifetimes were also determined for bacteriochlorophyll c in four of the solvents, and ranged from 2.7 ns in methanol to 7.6 ns in dimethylsulfoxide.
Nonphotochemical quenching (NPQ) of chlorophyll fluorescence plays an important role in the protection of plants against excessive light. Fluorescence quenching of the major light-harvesting complex (LHCII) provides a model system to study the mechanism of NPQ. The existence of both quenched and nonquenched states of LHCII has been postulated. We used time-resolved fluorescence and hydrostatic pressure to study differences between these states. Pressure shifts the thermodynamic equilibrium between the two states. The estimated volume difference was 5 mL/mol, indicating a local conformational switch. The estimated free energy difference was 7.0 kJ/mol: high enough to keep the quenched state population low under normal conditions, but low enough to switch in a controlled way. These properties are physiologically relevant properties, because they guarantee efficient light harvesting, while at the same time maintaining the capacity to switch to a quenched state. These results indicate that conformational changes of LHCII can play an important role in NPQ.
Because light is not required for catalytic turnover of the cytochrome b 6 f complex, the role of the single chlorophyll a in the structure and function of the complex is enigmatic. Photodamage from this pigment is minimized by its short singlet excited-state lifetime ( approximately 200 ps), which has been attributed to quenching by nearby aromatic residues ( Dashdorj et al., 2005). The crystal structure of the complex shows that the fifth ligand of the chlorophyll a contains two water molecules. On the basis of this structure, the properties of the bound chlorophyll and the complex were studied in the cyanobacterium, Synechococcus sp. PCC 7002, through site-directed mutagenesis of aromatic amino acids in the binding niche of the chlorophyll. The b 6 f complex was purified from three mutant strains, a double mutant Phe133Leu/Phe135Leu in subunit IV and single mutants Tyr112Phe and Trp125Leu in the cytochrome b 6 subunit. The purified b 6 f complex from Tyr112Phe or Phe133Leu/Phe135Leu mutants was characterized by (i) a loss of bound Chl and b heme, (ii) a shift in the absorbance peak and increase in bandwidth, (iii) multiple lifetime components, including one of 1.35 ns, and (iv) relatively small time-resolved absorbance anisotropy values of the Chl Q y band. A change in these properties was minimal in the Trp125Leu mutant. In vivo, no decrease in electron-transport efficiency was detected in any of the mutants. It was concluded that (a) perturbation of its aromatic residue niche influences the stability of the Chl a and one or both b hemes in the monomer of the b 6 f complex, and (b) Phe residues (Phe133/Phe135) of subunit IV are important in maintaining the short lifetime of the Chl a singlet excited state, thereby decreasing the probability of singlet oxygen formation.
With chlorophyll a-dipalmitoylphosphatidylcholine-liposomes, the absorption increases at 706 and 450 nm, and decreases at 660 and 420 nm, as the temperature is lowered. As the temperature is increased opposite changes are observed. A lipid phase change occurs at 34°. The pigment to lipid ratio is 1 to 5 in the liposome.
With chlorophyll a-soy bean lecithin-liposomes the absorption increases at 706, 680 and 440 nm, and decreases at 650 and 430 nm, as the temperature is lowered. As the temperature is increased, opposite changes are observed. A lipid phase change occurs at 26-27 °C. The pigment to lipid ratio is 1 to 13. The spectral change at 706 nm is identified with aggregated chlorophyll. The concentration of chlorophyll aggregate increases as the temperature is lowered, and decreases as the temperature is raised.
Fluorescence decay from chlorophyll a-soy bean lecithin-liposomes is biphasic. The lifetimes of freshly prepared liposomes are 121 ± 4 ps and 1400 ± 200 ps. The relative contribution of the fast and slow fluorescence components are modified by temperature. Heating results in an increase in both lifetimes, and an increase in fluorescence from the long lived component. These changes are interpreted as resulting from a decrease in energy transfer and concentration quenching. The origin of the biphasic fluorescence and spectral transformations in liposomes, and the possible relation between in vitro and in vivo picosecond fluorescence is discussed.
Absorption and fluorescence experiments on pheophytin and chlorophyll containing lipid bilayer vesicles are reported. Pheophytin aggregates on the vesicles are established from an additional red shifted band (at 695 nm) in the absorption spectrum. These aggregates contain pheophytin in an arrangement with the molecular planes of the porphyrin rings being parallel and cover about 10% of the vesicle surface. The lipid phase dissolves pheophytin up to a molar ratio of 15% above the lipid phase transition. This solubility limit decreases hardly on solidification of the lipid.
For chlorophyll a containing vesicles the aggregates are not observed in the absorption spectrum. The chlorophyll solubility is about equal to that of pheophytin. This suggests that the phase separa tion indicated from fluorescence measurements at temperatures below the lipid phase transition does not lead to the formation of strongly bound chlorophyll aggregates.
Electronic excitation energy transfer was studied for chlorophyll a in a solid solution of polystyrene by measuring the concentration quenching of quantum yield, polarization, and lifetime of fluorescence. The concentration quenching of the experimental fluorescence quantum yield is adequately described by Kelly and Porter's empirical formula (Proc. Roy. Soc., Lond. A 315, 149, 1970), and of polarization of fluorescence by the Jablonski theory (Acta Phys. Pol., 14, 295, 1955). With increasing concentration of chlorophyll a, the fluorescence peak at 672 nm (mainly monomer) is red-shifted, the intensity of the emission peak at ∼730 nm (mainly aggregate) relative to that at the shorter wavelength is increased. The [formula omitted] values, calculated by using total concentrations, for the emission at 672 nm and 730 nm are 73 ±2 Å and 45 ±1 Å, respectively. This may suggest that the chlorophyll monomers have a greater efficiency of energy transfer than the aggregates, which fluoresce at ∼730nm.
A quantitative analysis of fluorescence self-quenching of chlorophylls a and b in ether as well as chlorophyll a in lipid vesicles and liposomes has been carried out. It is demonstrated that concentration changes of the fluorescence quantum yield can be correctly described by a Förster-type excitation energy transfer process between chlorophyll molecules in the monomeric form if part of the transfers leads to energy degradation.
Es wird gezeigt, daß die auf einer Dipol-Dipol-Wechselwirkung beruhenden Vorgänge der zwischenmolekularen Energieübergabe sich auch im Rahmen der klassischen Quantentheorie quantitativ, ja sogar sehr anschaulich diskutieren lassen. Bei einer derartigen Erklärung sind alle Akte der Energieübergabe als solche mit Strahlung anzusehen. Nach der üblichen quantenmechanischen Auffassung ist i. allg. zwischen Übergabeakten mit und solchen ohne Strahlung zu unterscheiden: es scheint aber, daß die in einer Umgebung von genügend kleinem Radius der einzelnen angeregten Moleküle erfolgenden Elementarprozesse einheitlich als Akte ohne Strahlung aufgefaßt werden können.
Es werden auch bezüglich der die Übergabeprozesse begleitenden Löschprozesse Folgerungen gezogen
The fluorescence and force‐area behavior of chlorophyll a (Chl a) monomolecular films diluted with hydrocarbon on an aqueous subphase is reported. It is found that the diluent hexadecane, instead of conventional fatty alcohols, results in a substantial increase in the fluorescence yield of Chl a. The luminescence intensity varies nonmonotonically with respect to the intermólecular distance. A model for the mutual orientation of the chlorophyll pigments and the consequent energy transfer processes in terms of a Förster mechanism is proposed. For a random distribution of the Chl a molecules in the monolayer (k 2 = 2/3) an average value of 43° is calculated for the angle between the molecular planes and the water surface. This model yields a value of 23 Å for the Förster R 0. Also, a value of 58° is derived for the angle between the Q‐band transition moment and the line of intersection between the molecular plane and water surface. When a mixture of Chl a and Chl b in the monolayers is being excited at the absorption band of the latter pigment, typical fluorescence of Chl a is being detected. This sensitization process can also be interpreted in terms of the orientational model of the Chl molecules as presented in this work.
The three parameters, fluorescence polarization (P), relative quantum
yield (φ), and lifetime (τ) have been reinvestigated for
solutions of chlorophyll b in lecithin as a function of concentration.
All three show different concentration dependence, of which the
depolarization only, may be described by a simple single step energy
transfer. It is proposed that the self-quenching occurs when
nonfluorescent pairs of molecules are excited directly, and that the
reduction in lifetime is a result of energy transfer through the bulk
solution to such 'pairs' or other quencher. The origin of these 'pairs'
of molecules may be shown to be purely statistical. The apparatus used
for measurement of fluorescence lifetimes is described.
The concentration dependence of mean fluorescence lifetimes of chlorophyll a in methanol was studied in the concentration interval 3 × 10−6 − 7.3 × 10−5 mol l−1 at room temperature. The values found experimentally were corrected for reabsorption of fluorescence using the average molar absorptivity F derived from spectral properties of overlapping fluorescence and absorption spectra of chlorophyll a.
Site selection fluorescence and excitation spectra of chlorophyll-a in n-alkanes (octane, dodecane, hexadecane, eicosane) and polystyrene matrices were measured at low temperatures (5-30 K), using a pulsed dye laser tuned in the range 605 nm - 660 nm. The vibrational analysis of fluorescence quasilines gives very similar results for chlorophyll-a in matrices with different molecular chain lengths.
The zero-field fluorescence-detected triplet state magnetic resonance spectra have been obtained for the pyrochlorophyllide a—apomyoglobin complex at 2 K. The triplet state zero-field splittings and spin sublevel dynamics were detected on the resolved features of the structured low-temperature fluorescence. Structured fluorescence is not observed for pyrochlorophylide a in an organic matrix under identical conditions. These data are interpreted in terms of the local binding site of the pyrochlorophyllide a chromophore in the protein and the low-temperature conformation of the protein matrix.
Microemulsions, micelles, and vesicles are compared as media for membrane mimetic photochemistry. These systems solubilize, concentrate, compartmentalize, organize, and localize reactants; maintain proton and/or reactant gradients; alter quantum efficiencies; lower ionization potentials; change oxidation and reduction properties; change dissociation constants; affect vectorial electron displacements; alter photophysical pathways and rates; alter chemical pathways and rates; stabilize reactants, intermediates, and products; and separate products (charges). Formation of structures of microemulsions, micelles, and vesicles as well as substrate solubilization therein are summarized. Attention is focused on the utilization of microemulsions as reaction media. 72 references.
A modification of Förster's equation for the quantum yield of heterogeneous radiationless energy transfer improves its description of homogeneous concentration quenching, at the expense of a new parameter which can be related to the number of steps taken by the excitation in its random walk and which seems to be independent of concentration.
Fluorescence of aromatic molecules in solid solutions at room temperature and low temperature is quenched by addition of strong electron donors. The efficiency of quenching correlates well with calculated energetics for electron transfer (ET) from the added donors to the excited molecules, D + A* ..-->.. D/sup +/ + Aâ». The quencher (D) always had a substantially higher-lying excited state than did A to prevent electronic energy transfer (e.g., Foerster transfer). The A* fluorescence decreased exponentially with D concentration, being halved by addition of about 0.1 M D in cases with favorable energetics for electron transfer. These quenching measurements are interpreted as indicating electron transfer over distances up to 15 A (center to center). These maximum quenching distances are in excellent agreement with electron tunneling distances measured for ion-molecule reactions by pulse radiolysis. Formation of donor-acceptor complexes during sample preparation is a possible alternate interpretattion, but several features of the data provide evidence against this alternative.
The intensity of fluorescence of solutions of chlorophyll (either a or b) is sensibly independent of concentration, for values less than 2×10−3m. At higher concentrations, the fluorescence decreases with increasing concentration, conforming to the empirical equation, If,max/If=1+4300m2. The absorption spectrum of chlorophyll a is the same at 0.02m as it is for very dilute solutions. These results, as well as the observed temperature coefficient of the fluorescent intensity, indicate that the self-quenching of chlorophyll solutions is not due to the formation of non-fluorescent dimers nor to collisions of the second kind, but is very probably related to the resonance exchange of excitation (16, 17) between an excited and normal chlorophyll molecule.
Measurements of the fluorescence intensity of solutions containing mixtures of chlorophylls a and b indicate that chlorophyll b can sensitize the fluorescence of chlorophyll a, and that the fluorescence of chlorophyll b is more strongly quenched by a than it is by itself.
Chlorophyll a and oleyl alcohol form nearly ideal two‐dimensional solutions, up to chlorophyll mole fraction of at least 0.2. Chlorophyll is essentially insoluble, however, in stearyl alcohol monolayers at room temperature. Measurements of collapse pressure, area, absorption spectrum, and fluorescence spectrum of the monolayers confirm these conclusions. The fluorescence and absorption spectra of concentrated and diluted (in oleyl alcohol) chlorophyll films are compared. Measurable shifts in both absorption and emission demonstrate that chromophore—chromophore interactions are detectable in monolayers containing high chlorophyll concentrations.
Measurements have been made of the fluorescence spectra, lifetimes and quantum efficiencies and of the absorption spectra of several organic molecules in dilute solution. These have been used to test the validity of the theoretical relation between the radiative lifetime and the absorption intensity, and of the mirror-image relation between the fluorescence and absorption spectra. The former is found to be valid provided there is no major change in the nuclear configuration between the ground and excited states, but the latter is usually more sensitive to such changes. The effect of a change in nuclear configuration, which is particularly marked in the higher diphenylpolyenes, is to increase the radiative lifetime and to distort the spectral intensity distribution.
Optical and ODMR data for dimers of pheophytin a and b and chlorophyll a and b are presented. It is proposed that pheophytin a forms a parallel dimer arising from π-π interaction, binding being essentially different from that in the corresponding chlorophyll dimer. The dimer of phcophytin a is less stable (K ≈ 104 ol/mol) than that of chlorophyll a(K ≈ 106 ol/mol).
Studies have been made of the fluorescence and phosphorescence of solutions of all-trans and 15-15′-cis β-carotene. No significant differences were found in the light emission from these two isomers. At room temperature a weak green fluorescence is observed. It is proposed that both delayed fluorescence and phosphorescence occur in the delayed emission spectrum obtained at low temperature. A value of approximately 17,000 cm–1 is then obtained for the 0 â†� 0 triplet energy. The significance of this value in relation to other properties of β-carotene is discussed.
Intensity of fluorescence was determined as a function of chlorophyll concentration (in ethanol at 77°K) at the maxima of aggregate and monomer emission bands, 720 and 685 mμ, respectively. An expression, derived by relating chlorophyll concentration to the ratio of fluorescence at these two wavelengths, was found to be most consistent with experimental data when the aggregate was assumed to be a dimer. The fluorescence yield of the dimer in ethanol at 77°K was calculated and found to be 0.8. The lifetime of emission from this aggregated species corresponds to ∼10 -4 sec. Fluorescence from the chlorophyll monomer and aggregate, in vivo and in vitro, was measured as a function of temperature (from 293° to 77°K). In solution and in algae the activation energy for dimer emission is approximately 1 kcal, the Arrhenius constant is of the order of 10 4. The latter value reflects a low transition probability for dimer emission, suggesting origin in a triplet or non-binding singlet state.
A method for the determination of the absolute quantum yield of the fluorescence of solutions is described. This method uses the dipolar scattering of monochromatic light from glycogen solutions as a standard of unit quantum yield against which the fluorescence from a solution with the same apparent optical density for the exciting wavelength is compared. If both scattering and fluorescence are purely dipolar emissions, the linear polarization of the radiation emitted at right-angles to the direction of excitation is sufficient to characterize completely the spatial distribution of the radiation and together with the ratio of the intensities emitted in the same direction permits a comparison of the total emission from the two sources. The quantum yields of solutions of 28 substances, the fluorescence spectra of which range from 280 to 720 mμ, have been investigated and the results show satisfactory agreement with the most reliable values previously obtained by other methods.
Abstract— An electric field enhances the yield of fluorescence of chlorophyll in a liquid crystal solvent. The presented data suggest that the electric field effect is caused by the decrease in the efficiency of fluorescence quenching by ions. Quenching of each polarized component of fluorescence of chlorophyll molecules oriented by the liquid crystal matrix is different. The relative increase in fluorescence yield due to the applied electric field is stronger for a fluorescence component polarized parallel to the direction of liquid crystal orientation than for the perpendicular component.
The fluorescence of chlorophyll (Chl) a in 0.007–0.1% Triton X-100 was investigated by a phase-shift technique. The Chl a concentrations varied from 0.7 to 25 μM. Parallel measurements of fluorescence lifetime (τ) and quantum yield (ψ) were made. It was concluded that homogeneous energy transfer takes place at detergent concentrations above 0.025%: (i) the transfer between uniform molecules of the pigment solubilized in Triton X-100 micelles, when τ and ψ are constant; (ii) the transfer towards the quenching centers, resulting in a proportional decrease in τ and ψ. At a Triton X-100 concentration of about 0.025% the Chl a emission becomes heterogeneous. It is evident from the disproportional decrease in τ and ψ (greater in ψ than in τ) and also from the rise of the fluorescence at 730–750 nm. As the Triton X-100 concentration becomes lower than the critical one (0.021%), the number of micelles drops abruptly and Chl a forms colloid particles in the aqueous medium. This manifests itself as a decrease in τ and as a certain stabilization of ψ. Having analyzed the complex pattern of the τ/ψ ratio, we concluded that under these conditions more than 90% of Chl a is in a weakly fluorescent form (τ < 30 ps) and about 1% is in an aggregated state fluorescing at 732 nm with τ about 0.7 ns.
The absorption spectrum of chlorophyll a in aqueous methyl and butyl carbitol has been measured. The red-absorption maximum of chlorophyll dissolved in methyl carbitol (665 mμ) shifts to the red as the solvent is made increasingly aqueous (672 mμ in 30% methyl carbitol). Concurrently, the extinction coefficient decreases and fluorescence is lost. It has also been shown that only the non-fluorescent form of chlorophyll can form a colloid absorbing at 685 mμ in the presence of 6% dioxane.Butyl carbitol is more effective than methyl carbitol in keeping chlorophyll in a fluorescent form at high water concentrations, some fluorescence persisting when the solvent is 70–80% aqueous. Dioxane colloids prepared in 10% butyl carbitol exhibit an absorption maximum at 690 mμ, with a subsidiary maximum at 695 mμ. The 695-mμ absorption is considerably strengthened on standing. Thus, in these two solvent systems it is possible to prepare colloidal states of chlorophyll absorbing maximally at 672, 685, 690, and 695 mμ.
A microbeam flash photolysis apparatus has been developed for use with samples 50 to 250 mum square, and from 5 to several hundred microns thick. Triplets of chlorophyll a and b were observed in a number of solid solvents, including cholesterol, at room temperature without prior outgassing. In cholesterol the triplet yield decreased with increasing concentration according to the Stern-Volmer law, but the half life of the chlorophyll b triplet was 3 ± 0\cdot2 ms, and independent of concentration. Therefore, the excited singlet state but not the triplet is quenched by a concentration-dependent process. The half-quenching concentration of 2 x 10-3 M, corresponding to a mean intermolecular distance of 95 Å, points to quenching by inductive resonance. No triplets of chlorophyll appeared on flashing normal or etiolated plant leaves. Leaves treated with cationic detergent gave triplets in a yield of 15%, and exhibited increased fluorescence.
Changes in the fluorescence spectrum of chlorophyll a and b have been used to observe reversible changes that these substances undergo at high concentration and low temperature. In both cases the change is reflected in the appearance of an intense fluorescence band above 700 mμ. Absorption spectroscopy confirms the formation of reversibly aggregated species at temperatures below -100°.
Emission spectra of chlorophyll-a, dissolved in ethanol and various nonpolar solvents, were measured at 300°, 77°, and 4°K, in both dilute and concentrated solutions. For this purpose, a new technique utilizing fiber optics was employed. In dilute ethanolic solutions, the observed fluorescence bands emanate from chlorophyll-a monomers, solvated at the central magnesium. At 4°K the fluorescence yield in this solvent is 0.85. In highly concentrated ethanolic solutions, a low-temperature band occurs at about 724 nm, which may be attributed to solvated dimers, formed by a π—π interaction between the chlorophyll monomers. In nonpolar solvents, a low-temperature luminescence band is observed at about 755 nm. This emission is attributed to π—π* phosphorescence from unsolvated monomers. Also in nonpolar solvents, highly concentrated solutions have a weak band in the neighborhood of 733 nm at 77°K, which originates from chlorophyll dimers formed by interaction between the magnesium of one monomer with the cyclopentanone carbonyl of another. The 733-nm band is either π—π* phosphorescence or fluorescence. In CCl4 and benzene the dimers are believed to be unsolvated. At room temperature unsolvated dimers fluoresce feebly at 713 nm. Cooling dilute solutions of chlorophyll a in benzene and 1,2-dichloroethane causes dimer formation, but this is less in CCl4 and absent in ethanol. A calculation of the upper limit for the rate constant for intersystem crossing to the triplet state, in ethanol, gives a value of 9.0×106/sec.
The fluorescence yields and lifetimes of chlorophyll-a-in lipid liposomes and vesicles have been measured in an attempt to understand the light harvesting mechanism of photosynthesis. Concentration quenching of the fluorescence was observed in all systems, the extent depending on the lipid used. The system having the highest half-quenching concentration (7.0 × 10−2 molal) was 3:1 mole to mole mixture of monogalactosyl diglyceride and digalactosyl diglyceride.
By means of the Langmuir-Blodgett technique, monomolecular layers
containing chloroplast pigments and lipids have been prepared and
transferred from a water surface to solid substrates for spectroscopic
study. The absorption spectra of such monolayers of pure chlorophyll a
and b have been characterized on several different surfaces. Phytol and
M2+-1\colon 1 stearate-oleate have been used as inert,
transparent layer diluents for the chlorophylls to enable the effect of
pigment concentration on the absorption and fluorescence spectra to be
studied. Self-quenching of the fluorescence of both chlorophylls has
been observed and the chromophore separation at the half-quenching
concentration is compared with similar data for monolayers on the
surface of water, as well as solid and solution systems. Energy transfer
between chlorophyll b and a within a monolayer has also been
investigated, and found to involve essentially irreversible transfer of
excitation energy from b to a. Experiments involving the quenching of
chlorophyll fluorescence by quinones in the monolayer leads to the
conclusion that the efficiency of quinone quenching is not enhanced at
high chlorophyll concentrations. The monolayer properties of the
photosynthetically important quinone, plastoquinone A, were
investigated, and it was found to form very unstable layers, unsuitable
in the present work.
Absorption spectra, triplet state and fluorescence properties of solutions of chlorophylls b and a and pheophytins b and a in lecithin have been studied over a range of concentration from 10-4 to 5 × 10-2 mol l-1. Energy transfer has been investigated for the pairs chlorophyll b to a, pheophytins b to a, and is adequately described by the Forster inductive resonance mechanism. We have also examined the self-quenching of fluorescence and triplet state formation for each of the four molecules. With the exception of pheophytin b, which forms aggregates at high concentrations, there were no observable quenchers and, in each case, the half quenching concentration is close to that at which energy transfer between like molecules occurs. Mechanisms for this self-quenching and the possible relevance of these models to photosystem II in photosynthesis are discussed.
The method of flash photolysis has been used for direct measurement of the quantum yields of triplet formation in dilute chlorophyll solutions at 23 ^circC. In ether solution, the triplet yields were found to be 0.64 and 0.88 for chlorophyll a and b, respectively. In very dry hydrocarbon solution, the yields decrease by at least a factor of 5. It is suggested that the low yields of fluorescence and intersystem crossing in dry solvents are due to dissociation of excited singlet dimer chlorophyll into ground state monomer species.
When chlorophyll, together with certain other amphiphilic substances, is adsorbed to particles of polyethylene plasticized by incorporation of tetradecane, it is maintained in monomeric or oligomeric forms with characteristic absorption and fluorescence spectra. The present work describes the properties of chlorophyll a on such particles in the presence of the three isomeric N-(pyridyl)myristamides, and of the similarly shaped but not basic compound myristanilide, in an effort to ascertain the structural factors governing associations of these species. Absorption and fluorescence spectra at room temperature are resolved into minimal sets of Gaussian components, and relations between the component sets are proposed. The positions of the component bands and their relative abundance are characteristic of the amide used. The 3- and 4-pyridyl isomers bind more strongly to chlorophyll, probably by ligation of the pyridine nitrogen to Mg of the pigment. The 2-pyridyl isomer and myristanilide bind more weakly, probably through the amide carbonyl group. The association of chlorophyll into species with characteristic absorption and fluorescence bands is promoted more strongly by the 3- and 4-isomers than by the 2-isomer and myristanilide, and probably involves hydrogen bonding to chlorophyll carbonyl groups. A possible manner of association of chlorophyll in the presence of N,N-dimethylmyristamide is also presented. By way of comparison, chlorophyll adsorbed with dodecylpyridinium bromide, which lacks a nucleophilic function, is mainly in the microcrystalline hydrate form absorbing near 740 nm.
Quenching of the fluorescence of chlorophyll a in monomolecular films by copper pheophytin a, a non‐fluorescent chlorophyll derivative, has been measured as a function of quencher concentration. For some measurements the monolayers consisted entirely of chlorophyll plus an admixture of quencher (0.001–0.2 mole fraction). For other measurements, the pigments were dissolved in monolayers of the inert diluent oleyl alcohol. The fluorescence quenching data were analyzed in terms of the inductive resonance transfer mechanism of Förster for the chlorophyll—quencher interaction. For the dilute layers, where a single‐transfer calculation is satisfactory, the range of the chlorophyll—quencher interaction in the Förster theory is 40 Å. This result is in fortuitously good agreement with values of 38–41 Å derived from optical absorption and emission spectra also measured in dilute monomolecular layers. For the undiluted monolayers, where interactions among chlorophyll molecules are much more important, the single‐transfer model is not applicable. The fluorescence quenching data have been analyzed in terms of diffusion of localized excitations. Values of approximately 13 Å for the range of the chlorophyll—quencher interaction and 20–23 Å for the chlorophyll—chlorophyll interaction were derived from this analysis. These results are to be compared with values of 11–12 Å and 18 Å, for the two interaction ranges, respectively, calculated from the optical properties. It is pointed out that a collective excitation representation would be more appropriate for describing the undiluted monolayers.
An integrating sphere has been adapted to the measurement of the quantum yield of the fluorescence of solutions of pigments. The intensities of the fluorescence and of the absorbed light were determined separately with a thermopile.
Chlorophylls a and b dissolved in various solvents have been studied with this apparatus over a range of concentrations, and the measured yields corrected for autoabsorption. The yield for chlorophyll a is 0.25 and is in general independent of solvent and excitation wavelength. The yield for chlorophyll b is 0.11 for ether solutions and 0.06 for methanol solutions. The absolute fluorescent yields of solutions of fluorescein, eosine, magdala red, and rubrene were also measured. These results are in reasonable agreement with the published values of the yields for the same compounds.
An apparatus has been constructed which permits observation of the fluorescence of pigment molecules spread in monomolecular films on aqueous surfaces. It is found that while the emission from pure chlorophyll films is strongly concentration quenched, relative fluorescence yield increases nearly 1000‐fold when the chromophores are effectively separated by a two‐dimensional diluent. Factors affecting the efficacy of several diluents are discussed. The emission spectrum of diluted chlorophyll a monolayers is presented. Measurements of relative yield as a function of pigment concentration in mixed films containing chlorophyll and oleyl alcohol are consistent with a model involving energy migration to nonfluorescent centers in the monolayer. The present results, however, cannot demonstrate the quantitative validity of any specific mechanism for the migration process.
Measurements of stationary fluorescence polarization at wavelengths of excitation that yield limiting polarizations close to 1/2 and 1/7, respectively, permit the characterization of the rates of in−plane and out‐of‐plane rotation in aromatic compounds. Propylene glycol solutions of 1‐ and 2‐naphthylamine, some of their derivatives, anthracene, and indole show the expected dependence of the rate of rotation upon limiting polarization, but all of them additionally display on excitation at the red edge of the absorption, a decrease in the rotational rate, which then approaches the rate observed for pure out‐of‐plane rotations. Differential phase measurements of the polarized components of the fluorescence of 1‐naphthylamine confirm this effect. After careful check of the effects of temperature and excitation wavelength upon the emission spectrum and the fluorescence lifetimes, it is concluded that the red‐edge rotational anomaly arises from the existence of out‐of‐plane transition moments in absorption and emission in this spectral region. The physical origin of the effects and their relation to other observed red‐edge phenomena are discussed.
The equations usually given relating fluorescence lifetime to absorption intensity are strictly applicable only to atomic systems, whose transitions are sharp lines. This paper gives the derivation of a modified formula 1/τ0=2.880×10−9n2〈ν̃f−3〉Av−1(gl/gu) ∫ εdlnν̃,which should be valid for broad molecular bands when the transition is strongly allowed.
Lifetimes calculated by this formula have been compared with measured lifetimes for a number of organic molecules in solution. In most cases the values agree within experimental error, indicating that the formula is valid for such systems. The limitations of the formula and the results expected for weak or forbidden transitions are also discussed.
The Raman excitation profile spectrum of the ν1 band of
trans-β-carotene has been obtained in the preresonance region (16
900-19 000 cm-1). Five features are found in the spectrum which are
interpreted as vibrational structure of a low-lying excited 1Ag state
with its origin near 17 230+/-100 cm-1. This corresponds to a 1Bu-1Ag
energy gap of 3470+/-100 cm-1, and may help account for the
nonfluorescence of β-carotene.
IN the primary process of plant photosynthesis it is generally accepted that efficient energy migration occurs between about 300 molecules of chlorophyll a, with subsequent light collection by a chemical trap. Solutions of chlorophyll in vitro, whether in fluid solvents1, monolayers2,3, multilayers4,5 or, as shown recently in our laboratory, in rigid matrices of PMMA and in bilayer lipid vesicles, exhibit the phenomenon of concentration quenching of the excited state at concentrations much lower than those which are present in the chloroplast. At a chlorophyll concentration of 10−1 M, which is comparable with that in the chloroplast, none of the in vitro systems has a fluorescent yield as high as is found in vivo, especially when the photochemical traps are closed. To try to understand the apparent absence of concentration quenching in vivo, we have re-examined its mechanism in vitro, and conclude that each chlorophyll molecule in the light-collecting system must be separated from other chlorophyll molecules, so as to prevent trap formation by orbital overlap and so that the minimum distance, averaged over all orientations in a random array, is 10 Å.
Several groups have introduced chlorophyll a into artificial bilayer membranes in an attempt to develop a model system for studying the behavior of chlorophyll in the photosynthetic membrane. In order to investigate the organization of chlorophyll in these model systems, mixed bilayer systems containing chlorophyll a and distearoylphosphatidylcholine under conditions of excess water have been studied by differential thermal analysis. The resulting data suggest a phase diagram for this system consisting of a double eutectic with formation of a thermodynamic compound of defined stoichiometry between chlorophyll a and phospholipid at temperatures below the liquidus. The phase diagram may be simulated to obtain thermodynamic parameters characteristic of the compound phase. It is apparent that the organization and intermolecular interactions of chlorophyll in a bilayer membrane can very widely depending on the temperature and composition of the system. In particular, phase separation can occur within the membrane over certain temperature ranges, resulting in an inhomogeneous system. Thus in interpreting the physical and spectroscopic properties of chlorophyll a in bilayer membranes, it is essential to consider the phase state of the membrane and the organization and environment of the chlorophyll in the particular phase.
The fluorescence of chlorophyll a solutions is quenched efficiently by oxidizing agents such as quinones, arylnitro compounds, olefinic nitro compounds, nitrosonaphthol, methyl red, oxygen and nitric oxide. It is also quenched weakly by certain amino-aromatic reducing agents. This list of quenchers and their relative efficiencies corresponds, strikingly, to the series of com-pounds which have been reported23 as inhibitors and retarders for polymerization reactions. Quantitatively, the quenching of these solutions corresponds to the modified21 Stern-Volmer equation I0/I = 1 + k1[Q] + k2[Q]2. Less exactly, but apparently within the limits of experimental error, these data conform to the following empirical, one-constant equation I0/I = 1 + k[Q] + 1/8 k2[Q]2.
This paper considers the primary light-induced electron-transfer (ET) processes in the reaction center of photosynthetic bacteria which involved ET from the electronically excited state of the bacteriochlorophyll a dimer (BChL)/sub 2/ to bacteriopheophytin (BPh) ((I.1)) and ET from BPh/sup -/ to ubiquinone (Q) ((I.2)). Ultrafast reactions I.1 and I.2, which are practically temperature independent over the range 4 to 300 K, cannot be accounted for in terms of low-temperature nuclear tunneling through a nuclear barrier. Two mechanisms for ultrashort, temperature-independent processes I.1 and I.2 were examined. The rate of the ET reaction (I.2) is considerably longer than characteristic medium-induced vibrational relaxation rates, so that process I.2 has to occur from a thermally equilibrated nuclear configuration of BPh/sup -/Q. Reaction I.2 is assigned to an activationless nonadiabatic ET process, the short lifetimes for this reaction stemming from a large value of the electronic coupling V approximately equal to 4 cm/sup -1/. We propose that the ultrafast reaction (I.1) occurs from a nonequilibrium nuclear configuration of the (BChl)/sub 2/*BPh initially excited state which is located above the crossing point of the nuclear potential surfaces for (BChl)/sub 2/*BPh and for (BChl)/sub 2//sup +/BPh/sup -/. Such a novel ET mechanism involves competition between ET and vibrational relaxation. A theory has been developed to handle this problem and applied to reaction I.1. A microscopic molecular scheme for the primary events of charge separation in bacterial photosynthesis is proposed, which rests on the optimization of the intramolecular distortions of the equilibrium nuclear configurations and the intermolecular spatial organization of the donor and the acceptor. The molecular scheme is successful in accounting for the qualitative and the quantitative features of the primary ET rates.
Knowledge of solvent effects on the redox properties of bacteriochlorophyll (BChl) and bacteriopheophytin (BPheo) is important for understanding their possible role(s) as intermediate electron acceptors in the primary photochemistry of photosynthetic bacteria. In the present study, an investigation of the electrochemical behavior of these compounds by cyclic voltammetry (CV) and cyclic differential pulse voltammetry (CDPV) in several aprotic solvents has shown that BChl aggregation and ligation interactions have a significant effect on its redox potentials. In methylene chloride, the one-electron reduction potential of BChl was found to shift positively by 200 mV to a value nearly identical with that of BPheo in the same solvent. The shift is most readily explained by the presence of BChl aggregates in this solvent. The one-electron oxidation potential is relatively unaffected by aggregation. In contrast, the formation of six-coordinate BChl in tetrahydrofuran (two molecules of solvent coordinated to the Mg atom of BChl) affects both the one-electron reduction and one-electron oxidation potential, with the greatest effect on the latter. Solvent effects on the redox properties of BPheo were found to be much smaller, a finding consistent with its inability to undergo aggregation and coordination interactions similar to those of BChl.
The photoexcited triplet state properties of chlorophyll aggregate systems in vitro were investigated by zero-field optically detected magnetic resonance (ODMR) spectroscopy at 2 K. Measurements of the triplet state zero-field splittings, over-all triplet lifetimes, and individual spin sublevel intersystem crossing rate constants were obtained for solutions of chlorophyll a and zinc-substituted chlorophyll a and for the covalently linked dimeric derivative of pyrochlorophyllide a. The triplet-state properties found for these systems are interpreted within the framework of the triplet exciton model to assess the applicability of the exciton approach in determining the geometry of chlorophyll aggregate systems and to evaluate structural features of chlorophyll systems proposed as models for in vivo chlorophyll.
The effect of several types of BChl interactions of its resonance Raman (RR) spectrum has been examined. In monomeric BChl species, ligation interactions at the central Mg atom produce significant changes in its spectrum and two structure-sensitive bands are identified, which are analogous to those in other porphyrins. Hydrogen-bonding interactions in monomeric BChl affect only the C=O modes, which are weakly in resonance at the excitation wavelength used here. In aggregated BChl species, large differences are observed in the RR spectra as compared to monomeric BChI for two of the three types of aggregates studied. These are the BChl pyrazine adduct (bifunctional ligand aggregate) and the BChl hydrate (hydrogen-bonded aggregate) absorbing at 845 nm. Both of these aggregates display RR spectra which are distinct from one another as well. The third type of BChl aggregate, that formed through coordination interactions of the acetyl or keto carbonyl group of one molecule with the Mg atom of another (self-aggregate), exhibits a spectrum which differs from monomeric BChl only in relative band intensities; no frequency shifts are observed. Additional results which are discussed include the identification of metal-sensitive and deuteration-sensitive bands. A comparison of the RR spectra of BChl a and its metal-free analogue, BPheo a, shows there are three bands which are sensitive to metalation in the region 1000-1800 cm-1. The fully deuterated spectrum of BChI is substantially different from the fully protonated.
The path of (Chl a 2HâO)/sub n/ hydrated chlorophyll a in its photoreaction with water is studied by means of time-resolved fluorescence and electron spin resonance techniques. It is shown that the observed ESR effects are primarily attributable to the weakly fluorescent aggregates of chlorophyll a dihydrate, (Chl a 2HâO)/sub n greater than or equal to 2/. By contrast, monomeric chlorophyll a hydrate in nonpolar solutions containing an excess of water is photochemically inactive and instead gives rise to comparatively strong fluorescence as well as delayed fluorescence characterized by oscillatory behavior, indicative of a charge-transfer feedback mechanism. The observation of reversible, light-induced ESR signals of aggregated radical cations, (Chl a-2HâO)/sub n greater than or equal to 2//sup +/., in the 10â»Â¹-10-s domain, five decades in time removed from the oscillatory process of monomeric chlorophyll ..cap alpha.. hydrate suggests the critical dependence of Chl a photochemical properties on the state of molecular aggregation. Evidence is obtained for the aggregation of hydrated Chl a as the dimer, (Chl a 2HâO)â, and multiples of the hexamer, (Chl a 2HâO)â. The conversion of light into electrochemical potential is quantified by a weak coupling limit treatment of nonadiabatic electron transfer in terms of measured effects of ESR lifetime lengthening and line-width narrowing. The observed differences in the optical and photocatalytic properties of (Chl a HâO)â, (Chl a-2HâO)/sub n greater than or equal to 2/, monomeric hydrated Chl a, and Chl a not complexed with water provide the rationale for proposing different stereospecific Chl a-HâO aggregates as models to account for the dramatic differences in the properties of P680, P700, and light-harvesting chlorophyll associated with P680. 9 figures.
Ten ammonium amphiphiles which possess three long-chain alkyl tails (C//1//2 or C//1//6) were prepared. They form clear aqueous dispersions upon sonication. Electron microscopy and light scattering experiments indicated the formation of huge bilayer aggregates except for one case. These bilayers undergo the characteristic crystal-to-liquid crystal-phase transition, as confirmed by differential scanning calorimetry (DSC) and by fluorescence depolarization of a diphenylhexatriene probe. Riboflavin, a water-soluble fluorescent probe, was shown to be trapped in bilayer vesicles of some triple-chain amphiphiles. Mixing of these bilayers with those of single-chain and double chain amphiphiles was examined by DSC and by absorption spectroscopy.