Aggregation of chlorophylls in monolayers. V. The effect of water on chlorophyll a and chlorophyll b in mono and multilayer arrays

The nature of the interactions between water molecules and monolayers and multilayers of chlorophyll a (Chl a), and monolayers and multilayers of Chl b. obtained by the Langmuir-Blodgett technique, is examined by infrared spectroscopy. Following deposition of the monolayer or multilayer of Chl a or Chl b onto a plate, repetitive scans showed some modifications in the infrared spectra which are interpreted as a reorganization of the molecules as some water molecules leave the array. Drying the sample further modifies the spectra, which indicates the departure of more tenacious water molecules. Putting the sample in a moist atmosphere does not restore the original spectrum. This is an indication of a nonreversibie reorganization in the chlorophyll array. The spectra of the monolayer of chlorophyll are much more complicated than those of the multilayer, owing to the nonintegrating effect of the monolayer, which reveals the perturbing effect of the different dielectric milieux on each functional group. On the basis of the analysis of the spectra and the information gathered from the surface pressure isotherms, a model is proposed for the monolayer arrangement at the air/water interface which implies two set of dimers of water per molecule of chlorophyll. One pair of dimers constitutes the water of the first kind and is composed of vapor-like dimers. This kind of water is situated between the porphyrin planes of chlorophyll molecules and is easily removed from the monolayer. The second pair of dimers is composed of water of the second and third kinds situated between the Mg atom of the chlorophyll molecules and the water of the subphase. The second kind of water is closest to the Mg atom and is the most difficult one to remove. The third kind of water is closest to the surface and its mobility is intermediate between that of water of the first kind and that of water of the second kind. Comparing the infrared spectra of a freshly prepared monolayers of Chl a with the resonance Raman spectra of intact chloroplasts (M. Lutz, Biochim. Biophys. Acta 460 (1977) 408), we notice great similarities. This is an indication that the model we propose for the monolayer of Chl a could play an important role in the chloroplast.     

Aggregation of chlorophylls in monolayers.

The interaction of dioxane vapor with monolayer and multilayers of chlorophyll has been studied using electronic and infarared spectroscopies. Our results indicate the formation of a complex implying the oxygene of the dioxanc molecules with the magnesiums of adjacent chlorophyll molecules. These results are consistent with the molecular orbital calculations, using the "free electron network" method done by Le Brech, Leblanc and Antippa [Chem. Phys. Letters 26(1974) 37-44].     

Aggregation of chlorophylls in monolayers. Part IV. The reorganisation of chlorophyll a in multilayer array

The nature of the interaction between the chlorophyll a molecules in multilayer arrays obtained by the Langmuir-Blodgett technique is examined by electronic and infrared spectroscopies. Following the deposition of the multilayers, we observed a blue shift with time in the electronic spectra. This effect is monitored by infrared spectroscopy. The intensity of the coordinated ketone band is decreased while the intensity of the free ketone band is increased. These modifications are explained by the reorganization of the chlorophyll a molecules from an organized to a less organized one. The influence of H2O, D2O and SO2 vapors on the chlorophyll a multilayers give some informations on the role of water molecules in the aggregation of chlorophyll a in this ordered system. From these observations, a model is proposed for the multilayer arrangement implying two molecules of water per molecule of chlorophyll a.     

Infrared study of human serum very-low-density and low-density lipoproteins. Implication of esterified lipid C=O stretching bands for characterizing lipoproteins

Fourier-transform infrared (FT-IR) spectroscopy was applied to examine human serum very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) in aqueous solution and in solid film for characterizing lipid components. On the basis of the FT-IR second-derivative spectra for standard samples of triglycerides, cholesterol esters and phospholipids, it was found that the band at 1746 cm(-1) for VLDL and the band at 1738 cm(-1) for LDL were mainly due to the unsaturated triglycerides and unsaturated cholesterol esters, respectively. The implications of ester C=O stretching bands are discussed.     

A theoretical and comparative CNDO study of chlorophylls a and b and their beryllium homologs

CNDO/II calculations of chlorophylls a and b and their beryllium homologs show that the 3d atomic orbitals of the metal atoms contribute significantly to the σ and π bonding energies. In both chlorophylls a and b replacement of magnesium by beryllium stabilizes the total molecular energy by 40 kcal/mol.     

Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).     

Chlorophyll b to chlorophyll a conversion precedes chlorophyll degradation in Hordeum vulgare L.

This study reveals by in vivo deuterium labeling that in higher plants chlorophyll (Chl) b is converted to Chl a before degradation. For this purpose, de-greening of excised green primary leaves of barley (Hordeum vulgare) was induced by permanent darkness in the presence of heavy water (80 atom % (2)H). The resulting Chl a catabolite in the plant extract was subjected to chemical degradation by chromic acid. 3-(2-Hydroxyethyl)-4-methyl-maleimide, the key fragment that originates from the Chl catabolite, was isolated. High resolution (1)H-, (2)H-NMR and mass spectroscopy unequivocally demonstrates that a fraction of this maleimide fragment consists of a mono-deuterated methyl group. These results suggest that Chl b is converted into Chl a before degradation. Quantification proves that the initial ratio of Chl a:Chl b in the green plant is preserved to about 60-70% in the catabolite composition isolated from yellowing leaves. The incorporation of only one deuterium atom indicates the involvement of two distinguishable redox enzymes during the conversion.     

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.     

Picosecond time-resolved fluorescence study of chlorophyll organisation and excitation energy distribution in chloroplasts from wild-type barley and a mutant lacking chlorophyll b.

Picosecond time-resolved fluorescence spectroscopy has been used to investigate the fluorescence emission from wild-type barley chloroplasts and from chloroplasts of the barley mutant, chlorina f-2, which lacks the light-harvesting chlorophyll a/b-protein complex. Cation-controlled regulation of the distribution of excitation energy was studied in isolated chloroplasts at the Fo and Fm levels. It was found that: (a) The fluorescence decay curves were distinctly non-exponential, even at low excitation intensities (less than 2 x 10(14) photons . cm(-2). (b) The fluorescence decay curves could, however, be described by a dual exponential decay law. The wild-type barley chloroplasts gave a short-lived fluorescence component of approximately 140 ps and a long-lived component of 600 ps (Fo) or 1300 ps (Fm) in the presence of Mg2+; in comparison, the mutant barley yielded a short-lived fluorescence component of approx. 50 ps and a long-lived component of 194 ps (Fo) and 424 ps (Fm). (c) The absence of the light-harvesting chlorophyll a/b-protein complex in the mutant results in a low fluorescence quantum yield which is unaffected by the cation composition of the medium. (d) The fluorescence yield changes seen in steady-state experiments on closing Photosystem II reaction centres (Fm/Fo) or on the addition of MgCl2 (+Mg2+/-Mg2+) were in overall agreement with those calculated from the time-resolved fluorescence measurements. The results suggest that the short-lived fluorescence component is partly attributable to the chlorophyll a antenna of Photosystem I, and, in part, to those light-harvesting-Photosystem II pigment combinations which are strongly coupled to the Photosystem I antenna chlorophyll. The long-lived fluorescence component can be ascribed to the light-harvesting-Photosystem II pigment combinations not coupled with the antenna of Photosystem I. In the case of the mutant, the two components appear to be the separate emissions from the Photosystem I and Photosystem II antenna chlorophylls.