Monomeric Chlorophyll Research Articles

PHOTO‐EXCITED TRIPLET STATE OF DIMERIC CHLOROPHYLL a IN 3‐METHYLPENTANE RIGID MATRIX AT 77 K STUDIED BY FLASH PHOTOLYSIS

Abstract— Flash photolysis studies of dimeric and monomeric chlorophyll a were carried out at 77 K. The triplet‐triplet absorption spectrum of the dimeric chlorophyll a in 3‐methylpentane at 77 K is interpreted as a sum of the spectra of chlorophyll a in the ground and triplet states. The dimeric chlorophyll a in the triplet state is considered to have the half‐excited structure at 77 K without photodisaggregation owing to high viscosity of the solvent.

ASSOCIATION OF CHLOROPHYLL WITH AMIDES ON PLASTICIZED POLYETHYLENE PARTICLES–III. UNUSUAL SPECTRA OF CHLOROPHYLL a WITH N‐METHYLMYRISTAMIDE*

Abstract—The absorption spectrum of chlorophyll a, adsorbed with the amphiphilic amide, N‐methylmyristamide. to particles of polyethylene swollen with tetradecane, is unusual in that the red band apparently consists of three main components, which by Gaussian deconvolution are located at 664.5, 678 and 687 nm. The last is very narrow, with a band width of only 5‐6 nm at half maximum. At low amide concentration, the 744 nm band of chlorophyll hydrate is also observed. Room temperature fluorescence is weak, and indistinctly resolved into bands. However, on gradual cooling to 80 K, the fluorescence intensifies greatly and becomes resolvable into at least eight bands. The circular dichroism spectrum of the red band region shows optical rotatory strength in two narrow bands at 677 and 686 nm which is enormous, compared to that of monomeric chlorophyll or even the 744 nm hydrate. It is suggested that cyclic oligomer structures, in which adjacent chlorophylls are linked through the amide group of N‐methylmyristamide, might be responsible for the spectral phenomena. Unsubstituted myristamide and N.N‐dimethylmyristamide do not produce these narrow‐banded phenomena with chlorophyll at all.

Temperature Dependence of Absorption Spectra of Chlorophyll a in Hydrocarbons over the Temperature Range 300–77 K. Enthalpy and Entropy Changes for the Formation of Dimeric Chlorophyll a

Abstract The absorption spectra of chlorophyll a in isopentane, 3-methylpentane, and methylcyclohexane solutions were studied over the temperature range 300–77 K. In those hydrocarbon solutions which were prepared using methanol treatment, chlorophyll a forms hydrated dimer at low temperatures. The dimer has an absorption peak at 695 nm at 160 K, which gradually shifts to 706 nm with decreasing temperature from 160 K to 77 K. The enthalpy change (–ΔH) for the formation of the dimer was obtained to be 41.0±0.8 kJ mol−1, independent of the solvent used. The entropy changes (–ΔS) were 113±8 J K−1 mol−1 for isopentane, 118±8 J K−1 mol−1 for 3-methylpentane and 128±8 J K−1 mol−1 for methylcyclohexane solutions. Comparative studies carried out of the solutions prepared without the methanol treatment suggest the existence of two kinds of hydrated monomeric chlorophyll a.

THE EFFECT OF COORDINATING LIGANDS ON THE TRIPLET STATE OF CHLOROPHYLL

AbstractUnder laser excitation at 457.9 and 514.5 nm, a frozen solution of chlorophyll a in n‐octane displays fluorescence peak maxima at 2K that may be assigned to two distinct monomeric chlorophyll species. Using zero‐field fluorescence‐detected magnetic resonance the triplet state properties of the two chlorophyll species have been assigned to the monoligated and biligated chlorophyll monomer in which water serves as the ligand coordinated to the magnesium metal center. These triplet state properties for chlorophyll in solution are then utilized in interpreting triplet state results for in vivo chlorophylls associated with the light harvesting chlorophyll protein complex. It is shown that the triplet state data are consistent with attachment of the chlorophyll molecule to the protein site with a single ligand coordinated to the chlorophyll metal center.

Ligand effects on the triplet state of chlorophyll

Laser excitation at 457.9 and 514.5 nm of a frozen solution of chlorophyll a in n-octane produces two fluorescence peak maxima at 2 K that may be assigned to two distinct monomeric chlorophyll species. Utilizing zero-field fluorescence-detected magnetic resonance the triplet state properties of the two chlorophyll species may be assigned to the monoligated and biligated chlorophyll monomer in which water serves as the ligand coordinated to the magnesium metal center.

Parameters of the Triplet State and Spectral Properties of the Monomeric Chlorophyll in Liposomes at -196°C

Parameters of the Triplet State and Spectral Properties of the Monomeric Chlorophyll in Liposomes at -196°C

Characterization of bacteriochlorophyll interactions in vitro by resonance Raman spectroscopy

Characterization of bacteriochlorophyll interactions in vitro by resonance Raman spectroscopy

Complete assignment of the carbon‐13 NMR spectrum of chlorophyl a

AbstractThe 13C NMR spectrum (at natural abundance) of monomeric chlorophyll α in acetone‐d6 has been recorded to re‐examine the assignments of the low field (aromatic‐olefinic) region of the spectrum. The assignments, made by the examination of the fully coupled spectrum and by the use of long‐range selective 1H decoupling (LSPD) with low‐power irradiation, were compared with those of the previous reports. The results of the present work clarify the ambiguities previously encountered in the assignment of the 10a‐ester, 7c‐propionyl, P‐2‐phytyl, 2b‐vinyl, γ‐ and β‐methine carbon atoms, as well as the β‐pyrrolic carbon‐6 and α‐pyrrolic carbons −16 and −17 of chlorophyll α. Reassignment of the three last carbons was found necessary. Knowledge of the chemical shifts of these carbon atoms was considered to be particularly valuable, as it yields relevant information on the delocalized π electron system which is crucial for the function of chlorophyll in photosynthesis.

Monomeric chlorophyll a enol : Evidence for its possible role as the primary electron donor in photosystem I of plant photosynthesis

The chlorophyll a (Chl a) special-pair model of the primary donor of photosystem I (P700) does not account in a completely adequate fashion for the magnetic resonance properties observed for P700(+). Moreover, P700 is at least 420 mV easier to oxidize than is Chl a in vitro. Neither Chl a dimer formation nor selective ligation of Chl a can account for this potential difference. Enolization of the Chl a ring V beta-keto ester results in a very different pi electronic structure. The Chl a enol can be trapped as a silyl enol ether. In addition, the enol analog 9-desoxo-9,10-dehydro-Chl a can be prepared. Both the trapped enol and its 9-H analog are approximately 350 mV easier to oxidize than Chl a. The ESR spectrum of the cation radical consists of a single 6.1-G gaussian line that is line narrowed relative to that of Chl a(+) in a manner similar to P700(+). Electron-nuclear double resonance (ENDOR) spectroscopy resolves only a 3.5-MHz hyperfine splitting for the 3-methyl-group. The remaining splittings are all less than 3.5 MHz. The second moment of the ESR line of fully (13)C-enriched 9-desoxo-9,10-dehydro-Chl a(+) agrees with that of [(13)C]P700(+) to within 10%. Application of the special-pair model to the [(13)C]P700(+) second-moment data yields a 100% error. Ab initio molecular orbital calculations on ethyl chlorophyllide a enol cation bear out the ESR and ENDOR data. We conclude that a monomeric Chl a enol model provides a better description of the magnetic resonance parameters and oxidation potential of P700 than a Chl a special-pair model.

A light-induced spin-polarized triplet detected by EPR in Photosystem II reaction centers

A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680 +, and the reduced intermediate pheophytin, I −, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3–5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a, however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.

Measurement of the midpoint potential of the pheophytin acceptor of photosystem II

Primary photochemistry in photosystem II (PSII) involves at least three components: (1) P680, the primary electron donor [ 11, which has been reported to be a chlorophyll dimer [2,3] or trimer [3] although recent evidence favors its identification as a monomeric chlorophyll species [4,5]; (2) Q, a special plastoquinone [6], which functions as a relatively stable acceptor (200-600 ps at room temperature when secondary electron transport is not blocked [7]); (3) Ph, a pheophytin molecule which functions as an intermediate electron carrier between P680 and Q. The involvement of pheophytin as an electron acceptor in PSI1 was initially demonstrated in [8-lo]. Reversible photo-reduction of pheophytin in PSI1 was observed when Q was chemically reduced. At the same time an unsplit EPR radical signal attributed to Phwas reported [lo]. Later it was shown that a split EPR signal could be photo-induced under some circumstances and this was attributed to the reduced Ph which was involved in an interaction with the semiquinone-iron form of Q [ 1 l] by analogy to the situation in photosynthetic bacteria [ 121. Similar EPR [S] and optical spectra at 200 K [ 131 were observed in a different PSI1 reaction center preparation. These spectra were also attributed to a pheophytin anion. Recent ENDOR measurements are consistent with the identification of the reduced intermediate acceptor in PSI1 as a pheophytin anion [ 141. Also a picosecond time-resolved optical spectrum of Phhas been reported in PSI1 particles in PSI1 particles in which Q is chemically reduced [ 1 s]. Strong evidence for the presence of an intermediate acceptor between P680 and Q has also come from the discovery of a reaction center triplet state in PSI1

Ligated chlorophyll cation radicals: Their function in photosystem II of plant photosynthesis

Magnesium tetraphenylchlorin, a synthetic model for chlorophyll, exhibits significant variations in the unpaired spin densities of its cation radicals with concomitant changes in oxidation potentials as a function of solvent and axial ligand. Similar effects are observed for chlorophyll (Chl) a and its cation radicals. Oxidation potentials for Chl --> Chl(+.) as high as +0.9 V (against a normal hydrogen electrode) are observed in nonaqueous solvents, with linewidths of the electron spin resonance signals of monomeric Chl(+.) ranging between 9.2 and 7.8 G in solution. These changes in electronic configuration and ease of oxidation are attributed to mixing of two nearly degenerate ground states of the radicals theoretically predicted by molecular orbital calculations. Comparison of the properties of chlorophyll in vitro with the optical, redox, and magnetic characteristics attributed to P-680, the primary donor of photosystem II which mediates oxygen evolution in plant photosynthesis, leads us to suggest that P-680 may be a ligated chlorophyll monomer whose function as a phototrap is determined by interactions with its (protein?) environment.

Excitation Energy Transfer among Chlorophyll a Molecules in Polystyrene: Concentration Dependence of Quantum Yield, Polarization and Lifetime of Fluorescence

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 R̅0 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 ∼ 730 nm.

Photo-Induced Electron Transfer in Chlorophyll Containing Liposomes

Abstract Liposomes (lipid, ᴅʟ-α-dipalmitoyl-phosphatidylcholine) containing chlorophyll a (ratio lipid to chlorophyll 30:1) exhibit an absorption maximum at 670 nm. Upon oxidation with iodine these liposomes yield a chlorophyll radical that shows a ESR signal with a line width (peak to peak) ⊿Y = ~0.1 G and a g-value of 2.0022, consistent with the presence of monomeric chlorophyll. Under anaerobic conditions, in the absence of acceptors, no light induced ESR signal is observed, whereas under aerobic conditions the chlorophyll free radical is generated in the light. Acceptors having access to the lipid membrane, like Fe3+ -pyrophosphate and methylviologen, give rise to chlorophyll radical formation, or, like quinones, form a semiquinone radical in the light. Non-permeable acceptors, such as ferricyanide, NADP, FMN and cytochrome c do not act as acceptors and no chlorophyll radicals can be produced by light. Furthermore, light dependent and completely reversible electron transfer from N,N,N′,N′-tetramethyl-p-phenylenediamine to ubiquinone can be demonstrated. Liposomes containing chlorophyll, therefore, can serve as a model system for photosynthetic electron transport.

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2°K

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2°K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are D = 0.02794 ± 0.00007 cm −1 , E = 0.00382 ± 0.00007 cm −1 , i.e. equal to those of monomeric chlorophyll a to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperature 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.

In vitro preparation and characterization of a 700 nm absorbing chlorophyll-water adduct according to the proposed primary molecular unit in photosynthesis

1. This study characterizes chlorophyll a-H 2O adducts in vitro in order to establish their generic relationship to the recently proposed [15, 18–20, 31] primary molecular adduct in photosynthesis. The effects of water titration and temperature on the absorption, fluorescence, excitation, and redox properties of the various in vitro chlorophyll a aggregate species are investigated. 2. From fluorescence measurements, we conclude that the driest chlorophyll a sample contains an equimolar amount of water. This conclusion is consistent with earlier experimental work [2, 3, 14, 17, 31], and clarifies the origin of the controversial [15] Katz model [14] of chlorophyll a-H 2O interactions. 3. With increasing water concentration or as the temperature is lowered below room temperature, the A-663 monohydrate chlorophyll a · H 2O (species absorbing at 663 nm) is favored at the expense of the A-678 anhydrous aggregate according to the equilibrium 2H 2O + chlorophyll a 2 ai 2 chlorophyll a · H 2O. Under excess water conditions, A-663 is converted to A-743 (chlorophyll a · 2H 2O) n . 4. On slow sample cooling to T ⪅ 200 °K, we observe the growth of A-700 at the expense of A-663. There is a direct correspondence between the increasing (decreasing) absorption by A-700 ( A-663) and increasing (decreasing) fluorescence at 720 nm (664 nm). 5. It is concluded that A-700 is most probably the dimer participating in the equilibrium 2 chlorophyll a · H 2O ai (chlorophyll a · H 2O) 2. The A-700 band consists of two exciton components (separated by ≈ 280 cm −1) that are interpretable in terms of the dimeric origin of A-700. 6. The deconvoluted A-700 absorption spectrum and the excitation spectrum of the 720 nm fluorescence are compared with the light-minus-dark spectra of P-700. 7. It is found that A-700 is reversibly bleached by I 2 ( E 0 = 0.54 V). The significance of this observation is discussed in terms of the redox properties of monomeric chlorophyll a and P-700.

Chlorophyll interactions and light conversion in photosynthesis.

Chlorophyll a is a molecule with an unusual combination of electron donor-acceptor properties. The Ring V keto C=O group can function as donor, the central Mg atom as acceptor. In the absence of extraneous nucleophiles, donor-acceptor interactions form chlorophyll dimers, (Chl.,), and oligomers, (Chl2),. With monofunctional electron donors, monomeric chlorophyll species Chl �9 L 1 and Chl �9 L 2 form. Bifunctional donors may cross-link chlorophylls through Mg atoms to form large polynuclear adduets of colloidal dimensions. Examination of the visible absorption spectra by computer deconvolution techniques indicates considerable similarity between bulk or antenna chlorophyll in the plant and (Chl2)n. Electron spin resonance studies suggest that ESR photo-signal I associated with the photosynthetic reaction center of photosynthetic organisms could arise in a special pair of chlorophyll molecules (Chl H20 Chl)- +. Introduclion There is universal agreement that chlorophyll is the primary photo-receptor in plants, but the details of the mechanisms whereby the energy of light quanta are made available for subsequent chemical purposes remain for the most part to be established. The term "chlorophyll" is generic for a small group of closely related substances invariably present in all organisms that carry out oxidation-reduction reactions with energy supplied by light. The principal chlorophylls are chlorophyll a (Chl a) and b (green plants, algae), bacteriochlorophyll (BChl, purple photosynthetic bacteria), and chlorobium chlorophyll (green photosynthetic bacteria). Chlorophyll q and c~ It, 2] (diatoms, marine brown algae) and chlorophyll d (marine red algae) are minor or auxiliary chlorophylls. Chlorophyll a is without exception present in all organisms that carry out photosynthesis with the evolution of molecular oxygen. As the most widely distributed and most important of the chlorophylls, the discussion here will for the most part concern chlorophyll a. The chlorophylls are cyclic tetrapyrroles (Fig. t) and thus belong to the family of porphyrin compounds, which have a very important role in biology as active participants in respiratory pigments, electron transport agents, and oxidative enzymes. The chlorophylls, however, differ in some important respects from the true porphyrins. Although the methyl, ethyl, vinyl and propionic acid side-chains in the chlorophylls are characteristic of porphyrins, the alicyclic Ring V, which contains a keto C=O function at position C-9, is unique to the chlorophylls. The

Emission spectra of chlorophyll-alpha in polar and nonpolar solvents.

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.

Solvate and dimer equilibria in solutions of chlorophyll

The spectrum of chlorophyll b in very dry hydrocarbon solvents exhibits two maxima, of equal intensity, at 641 and 659 nm which are attributed to the chlorophyll dimer, whilst the spectrum in presence of basic solvent, having a single maximum at 641 nm, is attributed to the monosolvate. Unsolvated monomeric chlorophyll is probably never present in significant amounts in solution and assignment of the 659 nm band to an n-π * transition is almost certainly incorrect. When the basic solvent is pyridine the constant for the equilibrium dimer + 2 solvent ⇌ 2 monosolvate is 1·0 ± 0·2 x 10 3 l. mole -1 .