Beata Myśliwa-Kurdziel

Understanding chlorophylls: Central magnesium ion and phytyl as structural determinants

Phytol, a C20 alcohol esterifying the C-17(3) propionate, and Mg2+ ion chelated in the central cavity, are conservative structural constituents of chlorophylls. To evaluate their intramolecular structural effects we prepared a series of metal- and phytyl-free derivatives of bacteriochlorophyll a and applied them as model chlorophylls. A detailed spectroscopic study on the model pigments reveals meaningful differences in the spectral characteristics of the phytylated and non-phytylated pigments. Their analysis in terms of solvatochromism and axial coordination shows how the central Mg and phytyl residue shape the properties of the pigment. Surprisingly, the presence/absence of the central Mg has no effect on the solvatochromism of (bacterio)chlorophyll pi-electron system and the hydrophobicity of phytyl does not interfere with the first solvation shell of the chromophore. However, both residues significantly influence the conformation of the pigment macrocycle and the removal of either residue increases the macrocycle flexibility. The chelation of Mg has a flattening effect on the macrocycle whereas bulky phytyl residue seems to control the conformation of the chromophore via steric interactions with ring V and its substituents. The analysis of spectroscopic properties of bacteriochlorophyllide (free acid) shows that esterification of the C-17(3) propionate is necessary in chlorophylls because the carboxyl group may act as a strong chelator of the central Mg. These observations imply that the truncated chlorophylls used in theoretical studies are not adequate as models of native chromophores, especially when fine effects are to be modeled.

Phytol as one of the determinants of chlorophyll interactions in solution

Optical absorption and fluorescence parameters of chlorophyll a and the phytol-free chlorophyllide a, as well as of their Mg-depleted derivatives, were compared in a series of organic solvents. In contrast to prevailing opinion, the spectral properties of chlorophyll are not indifferent to the removal of phytol. The electronic absorption spectra of chlorophyll a and chlorophyllide a differ and display a different dependence on the nature of the solvent, which cannot be explained solely by the location of a charged carboxylic group in the proximity of the π– electron system. In fact, measurements in media of varying basicity show that deprotonation of the free carboxylic group in chlorophyllide, i.e., the presence of a negative point charge near the macrocycle, has no effect on pigment absorption spectra. Analysis of the solvent effect on the QY energies in terms of solvent polarity reveals that the phytyl moiety perturbs the spectral features of chlorophyll, mainly due to its interactions with the pigment solvation shell. The phytyl residue might also be thus partly involved in controlling the central metal ligation in chlorophylls. This influence of phytol on the spectral features of chlorophyll should be taken into account when comparing the spectra in solution with various spectral forms of chlorophyll in vivo.

Syntheses and DNA binding of new cationic porphyrin–tetrapeptide conjugates

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet. We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins. Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.

The influence of structure and redox state of prenylquinones on thermotropic phase behaviour of phospholipids in model membranes.

Our study was aimed to investigate the significance of the isoprenoid side chain size as well as redox state of the quinone ring for interaction of two main classes of prenylquinones: plastoquinones (PQ) and ubiquinones (UQ) with lipid bilayers. By use of differential scanning calorimetry (DSC) we have followed the thermotropic behaviour of multilamellar vesicles prepared from dipalmitoylphosphatidylcholine (DPPC) upon incorporation of increasing amount (1.3-12 mol%) of quinone (quinol) molecules. Our studies reveal that as the side chain is shorter (from 9 to 2 isoprenoid units) the height of the calorimetric profiles is reduced and the temperature of the main transition of DPPC (T(m)) decreases (T(m)=39.4 degrees C for a sample with 12 mol% of PQ-2), and then increases up to 39.8 degrees C for PQ-1. For the samples containing quinols the effect is more pronounced even at lower concentration. The greater influence of the added prenylquinones on the pretransition demonstrates a stronger distortion of the DPPC packing in the gel state. It seems that this is the isoprenoid side chain length rather than the redox state of prenylquinones that determines their effectiveness in perturbation of thermotropic properties of lipid bilayer.

Effect of xanthophyll pigments on fluorescence of chlorophyll a in LHC II embedded to liposomes

Abstract A major light-harvesting pigment-protein complex of Photosystem II (LHC II) isolated from rye leaves was incorporated to egg yolk lecithin liposomes containing various amounts (0-0.6 mol % with respect to lipid) of xanthophyll pigments: violaxanthin, lutein or zeaxanthin. It appeared that the addition of xanthophylls increased the level of the chlorophyll a fluorescence emission despite the screening effect of additional pigments in the system. Another phenomenon documented by the 77 K fluorescence emission spectra was the xanthophyll-related desaggregation of LHC II within the lipid phase of liposomes. The analysis of the parameters of chlorophyll a fluorescence decay in comparison to steady state fluorescence emission let us conclude that the fluorescence reabsorption in aggregated LHC II was a major cause of the lower level of fluorescence emission in that state of antenna proteins. Chlorophyll singlet excitations quenching brought about by violaxanthin, lutein and zeaxanthin is also documented in liposomes containing incorporated LHC II and exogenous xanthophyll pigments, and in liposomes containing xanthophyll pigments and chlorophyll b .

Fluorescence Lifetimes and Spectral Properties of Protochlorophyllide in Organic Solvents in Relation to the Respective Parameters In Vivo

In this work, absorption and fluorescence spectra of protochlorophyllide (Pchlide), as well as its fluorescence lifetime, were investigated in organic solvents having different physical properties. The obtained Pchlide spectral features are discussed in relation to the parameters describing solvent properties (refractive index and dielectric constant) and taking into account the specific solvent‐Pchlide interaction. The correlation of Pchlide Qy and Soret absorption bands with solvent polarizability function ((n2 ‐ 1)/(n2 + 2)) has been found; however, the dispersion of the observed points was rather high. A small Stokes shift of a magnitude between 50 and 300 cm1 was found, which indicates low sensitivity of Pchlide to nonspecific solvation. The fluorescence decay of Pchlide was single exponential in all the investigated solvents, with the lifetime value ranging from 5.2 ns for dioxane to 3.5 ns for methanol. Dependence of the obtained fluorescence lifetimes on the solvent orientation polarizability, a parameter being the function of both refractive index and dielectric constant, was discussed. In water‐methanol mixtures, a further decrease of the fluorescence lifetime was observed, giving values of 2.9 ns for 25% methanol. Double‐exponential decay of Pchlide fluorescence was found for Pchlide in a solution of 15% methanol with the lifetimes of 4.5 ± 0.5 ns and 1.2 ± 0.3 ns and in pure water with the lifetimes of 2.5 ± 0.5 ns and 0.4 ± 0.1 ns. The obtained results are discussed in relation to spectroscopic properties of Pchlide in vivo.

Fluorescence Lifetimes of Protochlorophyllide in Plants with Different Proportions of Short-wavelength and Long-wavelength Protochlorophyllide Spectral Forms¶

Dark‐grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild‐type pea (Pisum sativum) and its light‐independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low‐temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild‐type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide650–656 form but also for the short‐wavelength Pchlide forms. A fast component in the range 0.3–0.8 ns and a slow component in the range 5.1–7.1 ns were present in all samples, but the values varied, depending on species. The long‐wavelength Pchlide650–656 form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short‐wavelength Pchlide628–633 form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein‐bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.

Molecular organization of antifungal antibiotic amphotericin B in lipid monolayers studied by means of Fluorescence Lifetime Imaging Microscopy

Amphotericin B (AmB) is a life-saving polyene antibiotic used to treat deep-seated mycotic infections. Both the mode of therapeutic action as well as toxic side effects are directly dependent on molecular organization of the drug. Binding of AmB to lipid monolayers formed with dipalmitoylphosphatidylcholine, pure and containing 40 mol% cholesterol or ergosterol, the sterols of human and fungi respectively, has been examined by means of Fluorescence Lifetime Imaging Microscopy. AmB emits fluorescence with the characteristic lifetimes dependent on actual molecular organization: tau(M2) < or = 10 ps and tau(M1) = 0.35 ns in the monomeric state, the emission from the S(2) and the S(1) states respectively and tau(D) = 14 ns and tau(A) = 3.5 ns in the form of a dimer and associated dimers respectively. Analysis of the Langmuir-Blodgett films reveals that AmB binds to the lipid membranes and to the cholesterol-containing lipid membranes preferentially in the form of associated dimers. The same form of AmB appears in the membranes containing ergosterol but additionally the monomers and dimers of the drug can be observed, which can severely affect molecular organization of the lipid membrane. The results are discussed in terms of selectivity of AmB towards the ergosterol-containing biomembranes of fungi.

Origin of Chlorophyll Fluorescence in Plants at 55–75°C¶

The origin of heat‐induced chlorophyll fluorescence rise that appears at about 55–60°C during linear heating of leaves, chloroplasts or thylakoids (especially with a reduced content of grana thylakoids) was studied. This fluorescence rise was earlier attributed to photosystem I (PSI) emission. Our data show that the fluorescence rise originates from chlorophyll a (Chl a) molecules released from chlorophyll‐containing protein complexes denaturing at 55–60°C. This conclusion results mainly from Chl a fluorescence lifetime measurements with barley leaves of different Chl a content and absorption and emission spectra measurements with barley leaves preheated to selected temperatures. These data, supported by measurements of liposomes with different Chl a/lipid ratios, suggest that the released Chl a is dissolved in lipids of thylakoid membranes and that with increasing Chl a content in the lipid phase, the released Chl a tends to form low‐fluorescing aggregates. This is probably the reason for the suppressed fluorescence rise at 55–60°C and the decreasing fluorescence course at 60–75°C, which are observable during linear heating of plant material with a high Chl a/lipid ratio (e.g. green leaves, grana thylakoids, isolated PSII particles).

Fluorescence Lifetimes Study of α-Tocopherol and Biological Prenylquinols in Organic Solvents and Model Membranes

Abstract We have found that for biological prenyllipids, such as plastoquinol-9, α-tocopherol quinol, and α-tocopherol, the shortest fluorescence lifetimes were found in aprotic solvents (hexane, ethyl acetate) whereas the longest lifetimes were those of ubiquinonol-10 in these solvents. For all the investigated prenyllipids, fluorescence lifetime in alcohols increased along with an increase in solvent viscosity. In a concentrated hexane solution, the lifetimes of prenylquinols considerably decreased. This contrasts with methanol solutions, which is probably due to the self-association of these compounds in aprotic solvents. We have also found a correlation of the Stokes shift of prenyllipids fluorescence with the orientation polarizability of the solvents. Based on data obtained in organic solvents, measurements of the fluorescence lifetimes of prenyllipids in liposomes allowed an estimation of the relative distance of their fluorescent rings from the liposome membrane surface, and was found to be the shortest for α-tocopherol quinol in egg yolk phosphatidylcholine liposomes, and increased in the following order: α-tocopherol in dipalmitoyl phosphatidylcholine liposomes < α-tocopherol < plastoquinol-9 < ubiquinol-10 in egg-yolk phosphatidylcholine liposomes.