Gibberellic Acid effects on greening in pea seedlings

The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly.     

Light intensity regulates the accumulation of the major light-harvesting chlorophyll-protein in greening seedlings

Etiolated pea (Pisum sativum [L.] cv Progress 9) and barley (Hordeum vulgare [L.] cv Boone) seedlings greened under either low (40 microeinsteins per square meter per second) or high (550 microeinsteins per square meter per second) intensity light were analyzed for chlorophyll (Chl) content and the levels of mRNA and protein for the major light-harvesting chlorophyll (Chl)-protein of photosystem II (LHC-II). Low intensity plants accumulated Chl more rapidly than high intensity plants. Both single radial immunodiffusion analysis and mild sodium dodecyl sulfate-polyacrylamide gel electrophoresis green gels showed that low intensity plants also accumulated LHC-II protein more rapidly than high intensity plants, following a kinetic pattern similar to the total Chl data. In contrast, LHC-II mRNA levels appeared to be independent of LHC-II protein levels although pea and barley LHC-II mRNA exhibited different light intensity responses. The absence of coordination between LHC-II mRNA and protein levels suggested that the biosynthesis of LHC-II in greening seedlings is not limited by mRNA. A correlation (better than the 0.01 significance level) between LHC-II protein accumulation and Chl accumulation was found for both pea and barley. The accumulation of LHC-II protein was not linked to the development of photosynthetic electron transport. These results and the similar effect of light intensity on Chl content and LHC-II protein levels suggested that the availability of Chl may limit LHC-II protein accumulation in greening seedlings.     

Assignment of spectral substructures to pigment-binding sites in higher plant light-harvesting complex LHC-II

The trimeric main light-harvesting complex (LHC-II) is the only antenna complex of higher plants of which a high-resolution 3D structure has been obtained (Kühlbrandt, W., Wang, D., and Fujiyoshi, Y. (1994) Nature 367, 614-621) and which can be refolded in vitro from its components. Four different recombinant forms of LHC-II, each with a specific chlorophyll (Chl) binding site removed by site-directed mutagenesis, were refolded from heterologously overexpressed apoprotein, purified pigments, and lipid. Absorption spectra of mutant LHC-II were measured in the temperature range from 4 to 300 K and compared to likewise refolded wild-type complex and to native LHC-II isolated from pea chloroplasts. Chls at different binding sites have characteristic, well-defined absorption sub-bands. Mixed occupation of binding sites with Chls a and b is not observed. Temperature-dependent changes of the mutant absorption spectra reveal a consistent shift of the major difference bands but an irregular behavior of minor bands. A model of the spectral substructure of LHC-II is proposed which accounts for the different absorption properties of the 12 individual Chls in the complex, thus establishing a first consistent correlation between the 3D structure of LHC-II and its spectral properties. The spectral substructure is valid for recombinant and native LHC-II, indicating that both have the same spatial arrangement of Chls and that the refolded complex is fully functional.     

Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.

The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.     

Structural intermediate in the photocycle of a BLUF (sensor of blue light using FAD) protein Slr1694 in a Cyanobacterium Synechocystis sp. PCC6803

Slr1694 in Synechocystis sp. PCC6803 is a family of blue-light photoreceptors based on flavin adenine dinucleotide (FAD) called BLUF (sensor of blue light using FAD) proteins, which include AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis. Illumination of dark-state Slr1694 at 15 degrees C reversibly induced a signaling light state characterized by the red shift in the UV-visible spectrum and by the light-induced Fourier transform infrared (FTIR) difference spectrum for structural changes of a bound flavin and apo protein. Illumination at the medium-low temperature (-35 degrees C) led to the red shift in the UV-visible spectrum despite some small difference in the light-induced changes. In contrast, the -35 degrees C illumination resulted in a completely different light-induced FTIR spectrum, in which almost all of the bands were suppressed with the exception of the bands for the change of C4=O bonding of the FAD isoalloxazine ring. The C4=O bands were induced at -35 degrees C with almost the same intensity, but the band frequency for the light state was upshifted by 6 cm(-)(1). The changes in frequency of the light-state C4=O band and in amplitude of other bands showed the same temperature dependence with a half-change temperature at approximately -20 degrees C. It was indicated that the light-induced structural changes of apo protein and FAD were inhibited at low temperature with the exception of the change in hydrogen bonding to the C4=O group. The light-induced formation of the FTIR bands was similarly inhibited by sample dehydration. We discussed the possibility that this constrained light state is a trapped intermediate state in the photocycle of Slr1694.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

Light induced structural changes of a full-length protein and its BLUF domain in YcgF(Blrp), a blue-light sensing protein that uses FAD (BLUF)

Blue-light sensing proteins that use FAD (BLUF) are members of a blue-light receptor family that is widely distributed among microorganisms. The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain. The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity. Light-induced structural changes for the signaling state formation were studied using the light-induced Fourier transform infrared (FTIR) difference spectroscopy of both the full-length YcgF protein (YcgF-Full) and its BLUF domain (YcgF-BLUF). YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics. The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum. These bands were assigned to the light-induced structural changes of the protein. However, the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF. Furthermore, the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands. The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.     

Amino acid composition of the 9 kDa phosphoprotein of pea thylakoids

The 9 kDa phosphoprotein of pea thylakoids was isolated by electroelution from SDS-polyacrylamide gels and its amino acid composition determined. The result is at variance with the amino acid compositions predicted from published nucleotide sequences of the genes for apocytochrome b-559 and for CFo subunit III. The amino acid composition of the 9 kDa phosphoprotein resembles that of the 25 kDa light-harvesting chlorophyll a/b protein (LHC-II). We propose that the 9 kDa polypeptide is a chlorophyll-binding protein of photosystem II, that it functions as a link in excitation energy transfer between LHC-II and the reaction centre, and that its phosphorylation regulates excitation energy distribution by means of mutual electrostatic repulsion between itself and phosphorylated LHC-II.     

Light-Induced Changes in the Fluorescence Yield of Chlorophyll a In Vivo: I. Anacystis nidulans

he fluorescence yield of chlorophyll a in dark adapted Anacystis nidulans undergoes a slow change with continuous illumination. After the completion of the initial fast transient, the fluorescence yield rises from the level S to a plateau M within a minute, declining only after prolonged illumination. Both normal and 1,1-dimethyl-3(3'4'-dichloro)-phenylurea (DCMU)-poisoned Anacystis are capable of these changes. In normal Anacystis, the slow increase in the fluorescence yield (S --> M) requires light absorbed in system II while light absorbed in system I is ineffective. In DCMU-poisoned Anacystis, however, these changes are also promoted by light absorbed in system I. Addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), a photophosphorylation uncoupler acting near the photosynthetic electron transport chain, abolishes the rise from S to M in normal but has no effect in the DCMU-poisoned system. Phlorizin, a phosphorylase inhibitor, has very little effect. These results suggest that the light-induced variation in the fluorescence yield is related to the conformational changes which accompany photophosphorylation. The fluorescence yield of the auxiliary pigment phycocyanin remains constant throughout the interval of the light-induced changes in the fluorescence yield of chlorophyll a. Consequently, the fluorescence spectrum of the alga is variable on continuous illumination.     

Immunological Quantification of Proteins Related to Light-Harvesting Chlorophyll a/b Protein Complexes of the Two Photosystems in Rice Mutants Totally and Partially Deficient in Chlorophyll b

Ten rice chlorina mutants of Type I, which totally lack chlorophyllb and hence are unable to synthesize light-harvesting chlorophylla/b protein complexes of photosystem II (LHC-II), containedmRNA for proteins related to LHC-II. Immunoblotting with anantiserum, which had been raised against the 24 and 25 kDa apoproteinsof LHC-II and found to cross-react with the 26 kDa protein ofLHC-II and the 20 and 21 kDa apoproteins of light-harvestingchlorophyll a/b protein complexes of photosystem I (LHC-I),revealed that all the five proteins related to LHC-Iand LHC-IIwere present in reduced amounts in the Type I mutants. ThreeType HA mutants, which have a chlorophyll a/b ratio of 10, weremore abundant in the apoproteins, while three Type IIB mutantswith the ratio of 15 were heterogeneous in terms of the apoproteincontent. All the chlorina mutants contained less P700 comparedwith the wild type rice, but were relatively more abundant inthe LHC-I proteins than the LHC-II proteins. The results showthat all the rice chlorina strains are mutants of chlorophyllb synthesis and the deficiency of chlorophyll b differentlyaffects accumulation of the apoproteins of LHC-I and LHC-II.To balance light absorption between the two photosystem, lossof LHC-II is partly counter-balanced by a decrease in the numberof PSI complexes in the mutants. (Received January 21, 1988; Accepted April 28, 1988)     

Chloroplast Response in Dunaliella salina to Irradiance Stress (Effect on Thylakoid Membrane Protein Assembly and Function)

The chloroplast response in the green alga Dunaliella salina to irradiance stress was investigated. Cells were grown under low light (LL) at 100 [mu]mol photons m-2 s-1 or high light (HL) at 2000 [mu]mol photons m-2 s-1 incident intensity. LL-grown cells had a low chlorophyll (Chl) a/b ratio, an abundance of light-harvesting complex II proteins (LHC-II), and a large Chl antenna size. HL-grown cells had a higher Chl a/b ratio, relatively fewer LHC-II, and a small Chl antenna size. The more abundant higher molecular mass subunits of the LHC-II (approximately 31 kD) were selectively depleted from the thylakoid membrane of HL-grown cells. Light-shift experiments defined the kinetics of change in the subunit composition of the LHC-II and suggested distinct mechanisms in the acclimation of thylakoids to HL or LL conditions. The results showed that irradiance exerts a differential regulation on the expression of various Lhcb genes. The specific polyclonal antibodies used in this work, raised against the purified LHC-II, cross-reacted with a polypeptide of approximately 20 kD in HL-grown samples. In this work we examined the dynamics of induction of this novel protein and discuss its function in terms of a chloroplast response to the level of irradiance.     

Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fourier transform infrared difference spectroscopy shows no evidence for an enolization of chlorophyll a upon cation formation either in vitro or during P700 photooxidation

Molecular changes associated with the photooxidation of the primary electron donor P700 in photosystem I from cyanobacteria have been investigated with Fourier transform infrared (FTIR) difference spectroscopy. Highly resolved signals are observed in the carbonyl stretching frequency region of the light-induced FTIR spectra. In order to assign and to interpret these signals, the FTIR spectra of isolated chlorophyll a and pyrochlorophyll a (lacking the 10a-ester carbonyl) in both their neutral and cation states were investigated. Comparison of the redox-induced FTIR difference spectra of these two model compounds demonstrates that upon chlorophyll a cation formation in tetrahydrofuran the 7c-ester carbonyl is essentially unperturbed while the 10a-ester carbonyl is upshifted from 1738 to 1751 cm-1. For the 9-keto group, the shift is from 1693 to 1718 cm-1 in chlorophyll a and from 1686 to 1712 cm-1 in pyrochlorophyll a. The 1718-cm-1 band in the difference spectrum of chlorophyll a is thus unambiguously assigned to the 9-keto carbonyl of the cation. Comparison of the light-induced FTIR difference spectrum associated with the photooxidation of P700 in vivo with the difference FTIR spectrum of chlorophyll a cation formation leads to the assignment of the frequencies of the 9-keto carbonyl group(s) at 1700 cm-1 in P700 and at 1717 cm-1 in P700+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-modulated exposure of the light-harvesting complex II (LHCII) to protein kinase(s) and state transition in Chlamydomonas reinhardtii xanthophyll mutants

Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

Molecular changes following oxidoreduction of cytochrome b559 characterized by Fourier transform infrared difference spectroscopy and electron paramagnetic resonance: photooxidation in photosystem II and electrochemistry of isolated cytochrome b559 and iron protoporphyrin IX-bisimidazole model compounds.

The vibrational infrared absorption changes associated with the oxidation of cytochrome b559 (Cyt b559) have been characterized. In photosystem II (PS II) enriched membranes, low-potential (LP) and high-potential (HP) Cyt b559 were investigated by light-induced FTIR difference spectroscopy. The redox transition of isolated Cyt b559 is characterized by protein electrochemistry. On the basis of a model of the assembly of Cyt b559 with the two axial Fe ligands being histidine residues of two distinct polypeptides, each forming a transmembrane alpha-helix [Cramer, W.A., Theg, S.M., & Widger, W.R. (1986) Photosynth. Res. 10, 393-403], the bisimidazole and bismethylimidazole complexes of Fe protoporphyrin IX were electrochemically oxidized and reduced to detect the IR oxidation markers of the heme and its two axial ligands. Major bands at 1674/1553, 1535, and 1240 cm-1 are tentatively assigned to nu 37 (CaCm), nu 38-(CbCb) and delta (CmH) modes, respectively; other bands at 1626, 1613, 1455, 1415, and 1337 cm-1 are assigned to porphyrin skeletal and vinyl modes. Modes at 1103 and 1075/1066 cm-1 are assigned to the 4-methylimidazole and imidazole ligands, respectively. For the isolated Cyt b559, it is shown that both the heme (at 1556-1535, 1337, and 1239 cm-1), the histidine ligands at 1104 cm-1 and the protein (between 1600 and 1700 cm-1 and at 1545 cm-1) are affected by the charge stabilization. The excellent agreement between model compounds and isolated Cyt b559 reinforces the validity of the model of a heme iron coordinated to two histidine residues for Cyt b559. A differential signal at 1656/1641 cm-1 is assigned to peptide C = O mode(s). We speculate that this signal reflects the change in strength of a hydrogen bond formed between the histidine ligand(s) and the polypeptide backbone upon oxidoreduction of the cytochrome. In PS II membranes, the signals characteristic of Cyt b559 photooxidation are found at 1660/1652 and 1625 cm-1, for both the high- and low-potential forms. The differences observed in the amplitude of the 1660/1652-cm-1 band, at 1700 and 1530-1510 cm-1 in the light-induced FTIR difference spectra of Cyt b559 HP and LP, show that the mechanisms of heme oxidation in vivo imply different molecular processes for the two forms Cyt b559 HP and LP.     

Investigation of the molecular mechanism of the blue-light-specific excitation energy quenching in the plant antenna complex LHCII

Excitation of the major photosynthetic antenna complex of plants, LHCII, with blue light (470 nm) provides an advantage to plants, as it gives rise to chlorophyll a fluorescence lifetimes shorter than with excitation with red light (635 nm). This difference is particularly pronounced in fluorescence emission wavelengths longer than 715 nm. Illumination of LHCII preparation with blue light additionally induces fluorescence quenching, which develops on a minute timescale. This effect is much less efficient when induced by red light, despite the equalized energy absorbed in both the spectral regions. Simultaneous analysis of the fluorescence and photoacoustic signals in LHCII demonstrated that the light-driven fluorescence quenching is not associated with an increase in heat emission. Instead, a reversible light-induced conformational transformation of the protein takes place, as demonstrated by the FTIR technique. These findings are discussed in terms of the blue-light-specific excitation energy quenching in LHCII, which may have photoprotective applications.