Relevance of the diastereotopic ligation of magnesium atoms of chlorophylls in Photosystem I

The central magnesium (Mg) atoms of natural occurring tetrapyrroles such as chlorophylls (Chls) and bacteriochlorophylls (BChls) are typically five-coordinated, a fact which leads to the formation of diastereoisomers if the Mg-ligand bond is stable on the time scale of the observation method. This possibility has only been briefly addressed before in a CD-study of BChl c aggregates [T.S. Balaban, A.R. Holzwarth, K. Schaffner, J. Mol. Struct. 349 (1995) 183]. On the basis of the chlorophyll-protein complex photosystem I (PSI), which has recently been characterized by single crystal crystallography [P. Jordan, P. Fromme, H.T. Witt, O. Klukas, W. Saenger, N. Krauss, Nature 411 (2001) 909], we find that chlorophyll a molecules are much more frequently bound by the protein matrix from one side (anti) than the other one (syn) in a ratio of 82:14, which corresponds to a significant DeltaDeltaG value of 4.3 kJ/mol. Syn and anti denote the orientation of the Mg-ligand with respect to the 17-propionic acid esterified by phytol. Furthermore, by parallel sequence analysis we find that the binding sites for both syn and anti chlorophylls have been strongly conserved during evolution-a fact which stresses the nonrandom manner in which chlorophylls are bound by the apoprotein in antenna complexes, in order to exert efficiently their light harvesting function and energy funnelling. Most remarkably, all the syn chlorophylls are part of the inner core antenna system. Results from semiempirical quantum mechanical and detailed exciton coupling calculations allow us to speculate on the functional relevance of the diasteretopicity for PSI functioning.     

Uphill energy transfer in photosystem I from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>. Time-resolved fluorescence measurements at 77 K

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~?12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~?675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.     

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.     

Effect of some sterol-biosynthesis-inhibiting fungicides on the biosynthesis of polyisoprenoid compounds in barley seedings

The effect of five sterol-biosynthesis-inhibiting (SBI) fungicides, triadimefon, triarimol, diclobutrazol, tridemorph, and fenpropimorph on the germination, growth, and chloroplast pigment and sterol content of barley seedlings has been studied. Triadimefon, triarimol, and diclobutrazol at 250 microM depressed germination and growth, caused the accumulation of 14 alpha-methyl sterols, but had no effect on the formation of chlorophylls or carotenoids. Tridemorph and fenpropimorph at 250 microM had no effect on germination or the formation of chlorophylls and carotenoids but depressed growth and caused the accumulation of 9 beta,19-cyclopropyl sterols.     

Inhibition of porphobilinogenase by porphyrins in Saccharomyces cerevisiae

The biosynthesis of uroporphyrinogen III, the precursor of hemes, chlorophylls, corrins and related structures, is catalyzed by the porphobilinogenase system (PBGase), a complex of two enzymes, PBG-Deaminase (PBG-D) and Isomerase. Although the separate enzymes have been studied in some detail less work has been performed on the properties of the complex.In this study the kinetic behaviour of the enzyme PBGase in a normal yeast strain, D273-10B, and its derivative B231 has been investigated. Uroporphyrinogen formation was linear with time up to 2 hr at 37°C. The enzyme complex shows classical Michaelis-Menten kinetics. From the double reciprocal plots kinetic parameters were estimated for PBGase and PBG-D.Porphyrins were found to be competitive inhibitors with respect to porphobilinogen (PBG) and these compounds appeared to act as inhibitors by forming dead-end complexes with the free enzyme. 5-Aminolevulinic acid (ALA) also inhibited PBGase and this inhibition was overcome by addition of levulinic acid (2μM). These results indicate that ALA, is not an inhibitor but acts through its conversion into porphyrins which are the true inhibitors.     

Pathways and timescales of primary charge separation in the photosystem II reaction center as revealed by a simultaneous fit of time-resolved fluorescence and transient absorption

We model the dynamics of energy transfer and primary charge separation in isolated photosystem II (PSII) reaction centers. Different exciton models with specific site energies of the six core pigments and two peripheral chlorophylls (Chls) in combination with different charge transfer schemes have been compared using a simultaneous fit of the absorption, linear dichroism, circular dichroism, steady-state fluorescence, transient absorption upon different excitation wavelengths, and time-resolved fluorescence. To obtain a quantitative fit of the data we use the modified Redfield theory, with the experimental spectral density including coupling to low-frequency phonons and 48 high-frequency vibrations. The best fit has been obtained with a model implying that the final charge separation occurs via an intermediate state with charge separation within the special pair (RP(1)). This state is weakly dipole-allowed, due to mixing with the exciton states, and can be populated directly or via 100-fs energy transfer from the core-pigments. The RP(1) and next two radical pairs with the electron transfer to the accessory Chl (RP(2)) and to the pheophytin (RP(3)) are characterized by increased electron-phonon coupling and energetic disorder. In the RP(3) state, the hole is delocalized within the special pair, with a predominant localization at the inactive-branch Chl. The intrinsic time constants of electron transfer between the three radical pairs vary from subpicoseconds to several picoseconds (depending on the realization of the disorder). The equilibration between RP(1) and RP(2) is reached within 5 ps at room temperature. During the 5-100-ps period the equilibrated core pigments and radical pairs RP(1) and RP(2) are slowly populated from peripheral chlorophylls and depopulated due to the formation of the third radical pair, RP(3). The effective time constant of the RP(3) formation is 7.5 ps. The calculated dynamics of the pheophytin absorption at 545 nm displays an instantaneous bleach (30% of the total amplitude) followed by a slow increase of the bleaching amplitude with time constants of 15 and 12 ps for blue (662 nm) and red (695 nm) excitation, respectively.     

Evidence for two spectroscopically different dimers of light-harvesting complex I from green plants

A preparation consisting of isolated dimeric peripheral antenna complexes from green plant photosystem I (light-harvesting complex I or LHCI) has been characterized by means of (polarized) steady-state absorption and fluorescence spectroscopy at low temperatures. We show that this preparation can be described reasonably well by a mixture of two types of dimers. In the first dimer about 10% of all Q(y)() absorption of the chlorophylls arises from two chlorophylls with absorption and emission maxima at about 711 and 733 nm, respectively, whereas in the second about 10% of the absorption arises from two chlorophylls with absorption and emission maxima at about 693 and 702 nm, respectively. The remaining chlorophylls show spectroscopic properties comparable to those of the related peripheral antenna complexes of photosystem II. We attribute the first dimer to a heterodimer of the Lhca1 and Lhca4 proteins and the second to a hetero- or homodimer of the Lhca2 and/or Lhca3 proteins. We suggest that the chlorophylls responsible for the 733 nm emission (F-730) and 702 nm emission (F-702) are excitonically coupled dimers and that F-730 originates from one of the strongest coupled pair of chlorophylls observed in nature.     

The xanthophyll cycle is induced by light irrespective of water status in field-grown lavender (Lavandula stoechas) plants

The water relations, the photosynthetic capacity and the pigment content of leaves, i.e. chlorophylls, carotenes and xanthophylls, were analysed during the summer drought and recovery after autumn rainfalls in lavender ( Lavandula stoechas L.) plants grown in Mediterranean field conditions. Summer drought caused photoinhibition of photosynthesis and significant decreases in chlorophylls (by ca 75%), β -carotene (by ca 65%), and lutein and neoxanthin (by ca 50%), although their contents remained unaltered between predawn and midday, suggesting a progressive decrease in response to drought. In contrast, the levels of violaxanthin decreased from predawn to midday, giving rise to enhanced formation of zeaxanthin and antheraxanthin in high light. Zeaxanthin and antheraxanthin formation was not induced by water deficit. Although the levels of photosynthetic pigments were severely affected by water deficit, carotenoids decreased less than chlorophylls, which resulted in increased levels of carotenoids per unit of chlorophyll. We conclude that the enhanced formation of zeaxanthin in high light and the increased levels of carotenoids per unit of chlorophyll observed in water-stressed plants may help to avoid photoinhibitory damage to the photosynthetic apparatus.     

Die Hemmung der lichtinduzierten Chlorophyll-, Carotinoid- und Anthocyansynthese durch Äthanol

The Inhibition of the Light Induced Chlorophyll, Carotenoid and Anthocyan Synthesis by Ethanol. It is shown that already low concentrations of ethanol inhibit the light induced formation of pigments (chlorophylls, carotenoids, anthocyanins) in Raphanus-seedlings. The degree of inhibition depends on the ethanol concentration and rises with increasing incubation period. The inhibition of pigment synthesis is reversible, and immediately ceases when ethanol is removed. The formation of chlorophylls and anthocyanin is more repressed than that of carotenoids, and the formation of β-carotene to a higher extent than that of xanthophylls. It is concluded that ethanol inhibits pigment formation by inhibition of protein synthesis. These results indicate that ethanol and other lower alcohols cannot be used for the solubilizing of organic compounds when these are applied to plant tissues.     

Extinction coefficient for red-shifted chlorophylls: Chlorophyll d and chlorophyll f

Both chlorophyll f and chlorophyll d are red-shifted chlorophylls in oxygenic photosynthetic organisms, which extend photon absorbance into the near infrared region. This expands the range of light that can be used to drive photosynthesis. Quantitative determination of chlorophylls is a crucial step in the investigation of chlorophyll-photosynthetic reactions in the field of photobiology and photochemistry. No methods have yet been worked out for the quantitative determination of chlorophyll f. There is also no method available for the precise quantitative determination of chlorophyll d although it was discovered in 1943. In order to obtain the extinction coefficients (ε) of chlorophyll f and chlorophyll d, the concentrations of chlorophylls were determined by Inductive Coupled Plasma Mass Spectrometry according to the fact that each chlorophyll molecule contains one magnesium (Mg) atom. Molar extinction coefficient ε(chl f) is 71.11×10(3)Lmol(-1)A(707nm)cm(-1) and ε(chl d) is 63.68×10(3)Lmol(-1)A(697nm)cm(-1) in 100% methanol. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Self-aggregates of natural and modified chlorophylls as photosynthetic light-harvesting antenna systems: substituent effect on the B-ring.

Extramembranous light-harvesting antennae called 'chlorosomes' are the main sunlight-absorbing and energy-migrating systems in photosynthetic green bacteria. In a chlorosome, specific chlorophyllous pigments self-aggregate in hydrophobic environments surrounded by a lipid monolayer to form large oligomers. The self-aggregates of chlorosomal chlorophylls possessing a chlorin pi-system absorb sunlight and can emit near-infrared light, which is transferred to a bacteriochlorin pigment situated in the chlorosomal surface membrane. In vivo and in vitro self-aggregates of natural chlorosomal chlorophylls and their models have been investigated by electronic absorption analysis. Here their self-aggregation is reviewed from the viewpoint of substituent effect on the pyrrolic B-ring. Substituents at the 7- and 8-positions did not disturb the formation of their self-aggregates but affected their absorption bands.     

Nitrogen effects on proteins, chlorophylls and fatty acids during the growth of Arthrospira platensis

Spirulina platensis (=Arthrospira platensis) is a tunisian strain which has been isolated for the first time in Oued Essed (Sousse, Sidi Bou Ali). Biomass evolution, proteins, chlorophylls and fatty acids composition of this alga were monitored by varying nitrogen concentrations in the culture medium. Nitrogen stress was provoked by adding sodium nitrate (NaNO3) in the culture medium with concentrations varying from 0 to 5 g/l. Results obtained showed that nitrogen depletion increased total proteins and total chlorophylls. The addition of NaNO3 (5g/l) led to an increase of total fatty acids amounts and modify fatty acids composition. Optimal quantities of palmitic, gamma -linolenic and oleic acids were obtained with NaNO3 free-cultures. Thus, the tunisian strain has valuable biological substances, worthy to determine the optimal conditions for its propagation.     

Two equilibration pools of chlorophylls in the Photosystem I core antenna of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Summary Femtosecond transient absorption spectroscopy was applied for a comparative study of excitation decay in several different Photosystem I (PSI) core preparations from the green alga Chlamydomonas reinhardtii. For PSI cores with a fully interconnected network of chlorophylls, the excitation energy was equilibrated over a pool of chlorophylls absorbing at ∼683 nm, independent of excitation wavelength [Gibasiewicz et al. J Phys Chem B 105:11498–11506, 2001; J Phys Chem B 106:6322–6330, 2002]. In preparations with impaired connectivity between chlorophylls, we have found that the spectrum of chlorophylls connected to the reaction center (i.e., with ∼20 ps decay time) over which the excitation is equilibrated becomes excitation-wavelength-dependent. Excitation at 670 nm is finally equilibrated over chlorophylls absorbing at ∼675 nm, whereas excitation at 695 nm or 700 nm is equilibrated over chlorophylls absorbing at ∼683 nm. This indicates that in the vicinity of the reaction center there are two spectrally different and spatially separated pools of chlorophylls that are equally capable of effective excitation energy transfer to the reaction center. We propose that they are related to the two groups of central PSI core chlorophylls lying on the opposite sides of reaction center.     

Photosystem II: evolutionary perspectives

Based on the current model of its structure and function, photosystem II (PSII) seems to have evolved from an ancestor that was homodimeric in terms of its protein core and contained a special pair of chlorophylls as the photo-oxidizable cofactor. It is proposed that the key event in the evolution of PSII was a mutation that resulted in the separation of the two pigments that made up the special chlorophyll pair, making them into two chlorophylls that were neither special nor paired. These ordinary chlorophylls, along with the two adjacent monomeric chlorophylls, were very oxidizing: a property proposed to be intrinsic to monomeric chlorophylls in the environment provided by reaction centre (RC) proteins. It seems likely that other (mainly electrostatic) changes in the environments of the pigments probably tuned their redox potentials further but these changes would have been minor compared with the redox jump imposed by splitting of the special pair. This sudden increase in redox potential allowed the development of oxygen evolution. The highly oxidizing homodimeric RC would probably have been not only inefficient in terms of photochemistry and charge storage but also wasteful in terms of protein or pigments undergoing damage due to the oxidative chemistry. These problems would have constituted selective pressures in favour of the lop-sided, heterodimeric system that exists as PSII today, in which the highly oxidized species are limited to only one side of the heterodimer: the sacrificial, rapidly turned-over D1 protein. It is also suggested that one reason for maintaining an oxidizable tyrosine, TyrD, on the D2 side of the RC, is that the proton associated with its tyrosyl radical, has an electrostatic role in confining P(+) to the expendable D1 side.     

Changes in chlorophyll and carotenoid contents in radish (Raphanus sativus) cotyledons show different time courses during senescence

Changes of chlorophylls and carotenoids from green to yellow cotyledons of the radish ( Raphanus sativus ) were simultaneously and systematically analysed by high-performance liquid chromatography (HPLC) with photodiode array detection. Twenty-one components, seven chlorophylls and 14 carotenoids, were detectable. Seven chlorophylls and five carotenoids were identified from the results of HPLC analyses. Most chlorophyll species degraded during senescence, whereas carotenoids showed different behaviour in their metabolism depending on pigment species. For instance, during senescence, the contents of lutein and violaxanthin changed only slightly, β-carotene in 5-day-senescent cotyledons became 2.7 times higher than non-senescent leaves. Carotenoids of radish cotyledons were classified into three groups by their changes in concentration during senescence (increased, degraded and constant) and their roles discussed.     

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches. The potential role of chlorophyll molecules that are not bound to photosynthetic pigment-protein complexes ('free chlorophylls') or of other Mg-containing porphyrins in regulation of tetrapyrrole synthesis is also discussed.     

Chlorophyll transition dipole moment orientations and pathways for flow of excitation energy among the chlorophylls of the major plant antenna, LHCII

We have attempted in this work an assignment of the Qy dipole moment orientations for all the chlorophylls in the major plant antenna, light-harvesting complex II (LHCII). Information that has recently become available through a structural model of the LHCII, site-directed mutagenesis, and spectroscopy of both LHCII and CP29 has been evaluated to model the electronic excited state structure in the presence of chlorophyll-chlorophyll and chlorophyll-protein interactions. An assignment has been obtained which satisfactorily reproduces the polarized linear absorption characteristics. The assignment proposed has also been found to be adequate in reproducing the time scales of the energy transfer processes. The pathways for the flow of excitation energy among the chlorophylls of the complex have been suggested in the context of identity and orientation assignments.     

Etioplast and etio-chloroplast formation under natural conditions: the dark side of chlorophyll biosynthesis in angiosperms

Chloroplast development is usually regarded as proceeding from proplastids. However, direct or indirect conversion pathways have been described in the literature, the latter involving the etioplast or the etio-chloroplast stages. Etioplasts are characterized by the absence of chlorophylls (Chl-s) and the presence of a unique inner membrane network, the prolamellar body (PLB), whereas etio-chloroplasts contain Chl-s and small PLBs interconnected with chloroplast thylakoids. As etioplast development requires growth in darkness for several days, this stage is generally regarded as a nonnatural pathway of chloroplast development occurring only under laboratory conditions. In this article, we have reviewed the data in favor of the involvement of etioplasts and etio-chloroplasts as intermediary stage(s) in chloroplast formation under natural conditions, the molecular aspects of PLB formation and we propose a dynamic model for its regulation.     

Photophosphorylation, sensitized by chlorophills a and b, pheophytin and beta-carotene in a modelled system]

In aqueous solutions in air atmosphere chlorophylls a and b, pheophytin and beta-carotene adsorbed on aluminium oxide powder are capable of sensibilizing electron transfer from phosphate ions coupled with the formation of high energy bonds of adenosine phosphates. The highest activity of chlorophylls a and b and pheophytin is observed within the pH range of 7.5-7.8; that of beta-carotene--at pH 7.3-7.5.     

Excitonic interactions in wild-type and mutant PSI reaction centers

Femtosecond excitation of the red edge of the chlorophyll a Q(Y) transition band in photosystem I (PSI), with light of wavelength > or = 700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DeltaA between 635 and 665 nm, and four negative DeltaA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in photosystem 1. Exciton calculations, based on the recent PSI structure, allow an assignment of the DeltaA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. The subpicosecond transient absorption bands decay may reflect rapid charge separation in the PSI reaction center.