ANALYSIS OF THE COMPLEX BAND SPECTRUM OF P700 BASED ON PHOTOSELECTION STUDIES WITH PHOTOSYSTEM I PARTICLES

Abstract— The linear dichroism of the flash induced absorption changes of immobilized photosystem I reaction center particles from spinach chloroplasts has been investigated by means of the photoselection technique. Under flash excitation of predominantly β-carotenes at 500 nm, the photoinduced linear dichroism of the absorption changes has been measured in the spectral region from 620–720 nm. The most prominent feature in the spectrum of the dichroic ratio is the symmetrical pole at 687 nm. In parallel to all photoselection experiments, we recorded the light induced absorption changes of P700 under saturating flash excitation. A Gaussian deconvolution of this well-known difference spectrum of P700 has been attempted taking the additional features of the linear dichroism spectrum into account. From 670-720 nm, the best fit for both spectra was obtained by the following components:

• (1) 

  a disappearing wide band at 695.5 nm (σ= 200.0 cm⁻¹) attributed to the reduced special pair of chlorophyll a (Ch1- a );
• (2) 

  an appearing narrower band at 690 nm (σ= 120.0 cm⁻¹) with half the oscillatory strength of the former tentatively attributed to the non-oxidized moiety in the special pair; and
• (3) 

  a bathochromic bandshift centered at 688.4 nm attributed to the local electrochromic response of certain antennae Ch1- a molecules close to the primary electron donor.

The linear dichroism gives no evidence for any substructure within the absorption band of the reduced special pair.     

Sub-micro-second time-resolved absorption spectroscopy of a polar carotenoid analogue, 2-(all-trans-retinylidene)indan-1,3-dione; formation of the dication by direct triplet-excited sensitization

Sub-micro-second time-resolved difference absorption spectra of a polar carotenoid analogue, 2-(all-trans-retinylidene)indan-1,3-dione (hereafter, we will call RetInd), were recorded in tetrahydrofuran at room temperature upon anthracene-sensitized triplet excitation. In addition to the typical Tn <-- T1 absorption spectrum of anthracene followed by that of RetInd, a novel transient species, which peaked at 670 nm, was detected. The lifetime and the population of the 670 nm species was not affected by the presence of oxygen but was quenched by the cation scavenger, triethylamine. Therefore, we have identified this species as a "cation". The transient 670 nm species was not generated by direct photoexcitation of RetInd in the absence of a triplet sensitizer. Therefore, this species was not generated via the T1 species of RetInd but rather via an "invisible state" of RetInd, which is generated by direct energy or electron transfer from T1 anthracene. This proposed pathway was confirmed by a singular-value decomposition followed by a global fitting analysis. The "cation" of RetInd shows vibrational structure in its absorption spectrum, and its lifetime was determined to be 15 micros. Chemical oxidation of RetInd in 2,2,2-trifluoroethanol (dichloromethane) produced a broad absorption band around 880 (1013) nm, which could be transformed into a shoulder around 640 (675) nm upon addition of increasing amounts of the oxidant, FeCl3. The former absorption band can be assigned to a radical cation, while the latter to a dication. Because of the spectral similarity, the 670 nm species can be assigned to the dication, and the "invisible state" is ascribed to the radical cation of RetInd. This is the first direct evidence for the production of a dication of a biological polyene moiety generated in non-halogenated solution following anthracene-sensitized excitation.     

Trinuclear platinum(II) 4,6-diphenyl-2,2'-bipyridyl complex with bis(diphenylphosphinomethyl)phenylphosphine auxiliary ligand: synthesis, structural characterization, and photophysics

A trinuclear cyclometalated Pt(II) 4,6-diphenyl-2,2'-bipyridyl complex with bis(diphenylphosphinomethyl)phenylphosphine bridging ligand ([4-Ph(C--N--N)Pt](3)dpmp) has been synthesized and characterized. It exhibits a broad electronic absorption band from 400 to 600 nm because of its intramolecular Pt...Pt interactions that have been revealed by X-ray crystal structure analysis. This complex shows strong red emission in acetonitrile at room temperature and 77 K. The electronic and emission spectra exhibit concentration and temperature dependence. With increased concentrations, the UV band of the absorption spectrum gradually decreases and broadens, accompanied by an increase of the (1)[dsigma*,pi*] band between 400 and 600 nm. For emission spectra, the 550 nm band that originates from the mononuclear platinum(II) component gradually decreases with increased concentrations, while the band at approximately 700 nm that corresponds to the (3)[dsigma*,pi*] state increases. In addition, the UV-vis and emission spectra exhibit temperature and viscosity-dependence. The concentration-, temperature-, and viscosity-dependent characteristics indicate a conformational change of the complex arising from the rotation along the oligophosphine axis. This complex exhibits broad, positive, and strong transient difference absorption bands from the near-UV to near-IR spectral region. However, because of the increased ground-state absorption in the visible region, the nonlinear transmission of this trinuclear platinum complex decreases.     

Visible and near-infrared chiroptical gels containing electrochromic anthraquinone imide groups

A cholesterol-based organogelator bearing an anthraquinone imide(AQI) group was synthesized and characterized.It self-assembled into chiral gels in acetonitrile at low concentrations,which displayed a combination of electrochromic and chiroptical properties.Upon electrochemical reduction at -700 mV,the gel exhibited new absorption bands at around 820 nm corresponding toπ~*-π~*(SOMO→LUMO) transitions of the radical anion of AQI and strong negative Cotton effects in the same spectral region.With further reduction at -1000 mV,a new CD band with a negative Cotton effect in the range from 500 nm to 800 nm appeared concomitant with the variation of absorption spectrum.Thus, with the use of electrochromic AQI chromophore as a switch-responsive unit and the stable gel of compound N-[3β-cholest-5 -en-3-yl N-(2-aminoethyl) carbamate]anthraquinone-2,3-dicarboxylic imide as a chiral scaffold,a redox-triggered chiroptical switch operating in visible and near-infrared region was realized.     

ELECTROCHROMIC BAND SHIFTS OF CAROTENOID IN A BLUE-GREEN ALGA

Abstract— The excitation with a short flash of cells of a blue-green alga, Synechococcus sp., induced, besides photooxidation of cytochrome c -553 and P-700, small absorption changes of complex kinetics in the wavelength region between 450 and 570 nm. The absorption changes were resolved into two kinetic components different in their sensitivity to gramicidin D.
The ionophore-sensitive component (Gs), which rose very rapidly on flash illumination and decayed with a half-time of 3 ms, has spectral features indicating a red shift of carotenoid absorption bands. Gs was sensitive to valonomycin but not to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation rate of Gs was markedly slowed down in the presence of tri- n -butyltin chloride. Phenazine methosulfate induced a secondary slow rise following the initial rapid rise. A similar slow rise appeared in the dark-starved cells but disappeared on the addition of methyl viologen. It is concluded from these results that Gs is an electrochromic band shift of carotenoid responding to the electric field formed by the primary charge separation of the photosystem I reaction center and its decay is related to the proton translocation through a proton channel of the membrane.
The ionophore-resistant component rose and decayed with the half-times of 0.2 and 2 ms, respectively. Its difference spectrum suggests a blue band shift of carotenoid. The ionophore-resistant component was also insensitive to DCMU. However, this component may be related in some way to flash-induced electron flow, because the photoresponse was altered by dibromothymoquinone, bathophenanthroline and 2- n -heptyl-hydroxyquinoline- N -oxide or the dark starvation of cells, which were all effective in inhibiting the cytochrome c -553 reduction.     

ANALYSIS OF LASER-INDUCED FLUORESCENCE LINE SHAPE OF INTACT LEAVES: APPLICATION TO UV STRESS DETECTION

Abstract— In vivo laser-induced fluorescence spectra of intact leaves of healthy and UV-irradiated Salvia splendens plants excited at 337 nm by a nitrogen laser were recorded using an optical multichannel analyzer system. The spectra showed the typical fluorescence bands centered around 450, 530, 685 and 730 nm. Exposure to UV radiation changed the relative intensity values of these bands and their peak positions. The analysis of the acquired spectra in terms of a linear combination of Gaussian bands was carried out to determine accurately the peak positions and the relative intensity contribution of the various bands to the laser-induced fluorescence spectra on healthy and UV-treated plants of different age.
The results indicate that a curve-fitting analysis of the measured fluorescence spectra is a useful and sensitive method to discriminate the various band contribution to the whole leaf fluorescence spectrum. The comparison among blue-green and red-far-red fluorescence of leaves was also confirmed as an effective indicator of UV stress in plants.     

ACTION SPECTRA FOR LIGHT-INDUCED DE-EPOXIDATION AND EPOXIDATION OF XANTHOPHYLLS IN SPINACH LEAF*

Abstract— The action spectra for violaxanthin de-epoxidation and zeaxanthin epoxidation in New Zealand spinach leaf segments, Tetragonia expansa, were determined at equal incident quanta of 2·0 × 10¹⁵ quanta cm^-2 sec^-1. Precise action spectra were not obtained due to variable leaf activity. The de-epoxidation action spectrum had major peaks at approximately 480 and 648 nm. Blue light was slightly more effective than red light and little activity was observed beyond 700 nm. The epoxidation action spectrum showed major peaks at around 440 and 670 nm. Blue light was more effective than red light and light beyond 700 nm showed definite activity. The net result of de-epoxidation and epoxidation is a cyclic scheme, the violaxanthin cycle, which consumes O₂ and photoproducts. The action spectra indicate that the violaxanthin cycle is more active in blue than in red light and therefore could account for O₂ uptake stimulated by blue light. However, the violaxanthin cycle is not the pathway for O₂ uptake by photosynthetic system 1. It was suggested that the violaxanthin cycle may function as a pathway for the consumption of excess photoproducts generated in blue light or the conversion of these photo-products to other forms of energy.     

Time-resolved spectroscopy of energy transfer and trapping upon selective excitation in membranes of Heliobacillus mobilis at low temperature

Transient absorption difference spectra in the Qy absorption band of bacteriochlorophyll (BChl) g and in the 670 nm absorption band of the primary acceptor A0 in membranes of Heliobacillus mobilis (Hc. mobilis) were measured at 20 K upon selective excitation at 668, 793, 810, and 815 nm with a 5 nm spectral bandwidth. When excited at 793 nm, the spectral equilibration of excitations from shorter to longer wavelength-absorbing pigments occurred within 3 ps and mostly localized at the band centered around 808 nm. When excited at 668 nm, the excitation energy transfer from the 670 nm absorbing pigment to the Qy band of BChl g took less than 0.5 ps, and the energy redistribution occurred and localized at 808 nm as in the case of the 793 nm excitation. All of the excitations were localized at the long wavelength pigment pool centered around 810 or 813 nm when excited at 810 or 815 nm. A slower energy transfer process with a time constant of 15 ps was also observed within the pool of long wavelength-absorbing pigments upon selective excitation at different wavelengths as has been observed by Lin et al. (Biophys. J. 1994, 67, 2479) when excited at 590 nm. Energy transfer from long wavelength antenna molecules to the primary electron donor P798 followed by the formation of P+ took place with a time constant of 55-70 ps for all excitations. Direct excitation of the primary electron acceptor A0, which absorbed at 670 nm, showed the same kinetic behavior as in the case when different forms of antenna pigments were excited in the Qy region. This observation generally supports the trapping-limited case of energy transfer in which the excitations have high escape probability from the reaction center (RC) until the charge separation takes place. Possible mechanisms to account for the apparent "uphill" energy transfer from the long wavelength antenna pigments to P798 are discussed.     

PHOTOPHYSICS IN LIQUID CRYSTALS. FLUORESCENCE BEHAVIOR OF CHLOROPHYLL-a IN DODECYLCYANOBIPHENYL

Abstract— Fluorescence behaviour of chlorophyll- a in a liquid crystalline medium, dodecyl cyanobiphenyl, has been investigated as a function of temperature and concentration. At concentrations of 6 times 10^-4 M , emission in the Q-band region exhibited peaks in the crystal at 650,695 and 735 nm as well as the monomer band at 675 nm. Dilution studies indicate that these additional bands probably arise from aggregation of the chlorophyll.     

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 Pchlide(650-656) 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 Pchlide(650-656) 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 Pchlide(628-633) 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.     

聚氧乙烯－高氯酸锂配合物电致变色光窗的研究

以ＷＯ３为电致变色层、ＮｉＯｘ为离子贮存层、具有较好透光性的氧亚甲基连接聚氧乙烯／ＬｉＣｌＯ４，配合物为电解质制备了全固态电致变色光窗。电色窗在７００～９００ｎｍ之间的调制深度超过５０％。电色窗的电色能力随温度的升高及ＬｉＣｌＯ４盐浓度的增大而加强，并且电色窗具有较好的信息贮存能力。     

AQUEOUS AGGREGATES OF BACTERIOCHLOROPHYLL c AS A MODEL FOR PIGMENT ORGANIZATION IN CHLOROSOMES

Chlorosomes isolated from Chloroflexus aurantiacus were extracted with chloroform/methanol. The extract contained bacteriochlorophylls c and a and lipids, but was devoid of proteins. This crude extract spontaneously formed aggregates when a methanol solution was dispersed in aqueous buffer. The aggregates could be sedimented by ultracentrifugation and appeared in electron micrographs as stain-excluding bodies with diameters between 70 and 170 nm. The absorption spectrum is remarkably similar to that of intact chlorosomes with an absorption maximum of bacteriochlorophyll c at around 740 nm. The circular dichroism spectrum of the aggregate is also very similar to that of intact chlorosomes. A conservative (±) band centered at 740 nm confirms the highly aggregated state of bacteriochlorophyll c in both systems. Steady-state fluorescence studies showed that in the aggregate energy-transfer from bacteriochlorophyll c to a component emitting at 830 nm took place. When the aggregate was suspended in buffer saturated with 1-hexanol the 740 nm form of bacteriochlorophyll c was reversibly converted to a form with spectral properties resembling the monomer absorbing at 670 nm but still in an aggregated state. This form of bacteriochlorophyll c showed no circular dichroism signal.     

Peroxidative reactions attenuate oxygen effect on spectroscopic properties of isolated chloroplasts.

Steady-state absorption and fluorescence excitation spectra have been measured at 25 degrees C in order to elucidate the differences between isolated chloroplasts from pea (chilling-sensitive plant) and bean (chilling-tolerant plant) and their response to oxygen treatment. A weaker light harvesting in bean in comparison with pea chloroplasts is related to higher free fatty acids level and extended peroxidation activities of bean chloroplasts. Peroxidation of free fatty acids in bean chloroplasts results in an accumulation of oxygenated forms of fatty acids demonstrated by a large negative band around 400 nm in absorption difference spectra, while the excitation spectra are not significantly altered. Similar changes have been observed in the lipase-treated pea chloroplasts. In contrast, in both pea and bean chloroplasts exhibiting no peroxidation due to antimycin A treatment, oxygen induces a pronounced absorbance increase in the regions around 435, 470 and 674 nm indicating the chloroplast swelling. A decline of chlorophyll fluorescence excitation caused by oxygen, may result from a decrease in energy transfer from antennae complexes to chlorophyll species emitting at both 680 and 740 nm. The oxygen-induced changes are partially reversed upon restoration of anaerobic conditions. The presented data show for the first time, that in contrast to pea chloroplasts the peroxidation abolishes an oxygen-induced decrease in light harvesting in bean chloroplasts, i.e., a chilling-sensitive plant.     

ASSOCIATION OF CHLOROPHYLL WITH AMIDES ON PLASTICIZED POLYETHYLENE PARTICLES—I. N,N-DIMETH YLM YRISTAMIDE*

Abstract— Model systems have been prepared in which chlorophyll a (Chl a) and N.N-dimethylmyristamide (DMMA) are adsorbed together in various ratios to particles of polyethylene swollen with undecane. The adsorption is performed by equilibrating the particles with methanol-water solutions of increasing water content. Absorption spectra of the coated particles in viscous suspenions show sharp well-marked bands over much of the composition range examined. With the aid of second derivative spectra. the red absorption band has been resolved into three components. at 661.5. 674 and 680 nm. Fluoresccnce spectra have also been resolved into their principal components with some assistance from comparison with spectra of Chl in undecane solution containing DMMA. At room temperature (295 K) the resolvable components are of monomeric Chl at 670 nm. and of associated species at 681 and 725 nm. Fluorescence at 77 Kis of similar intensity but is distributed differently. in favor of longer-wave components. Corresponding to the 295 K components are emission bands at 675, 683–5 and 735 nm. Othcr components appear under certain conditions: at 695–700 nm when the Chl and DMMA conccntrations are both high, and at 705 nm whcn the ratio of DMMA to Chl is low. If DMMA is absent or at low concentration, much of the Chl exists as an aggregated form absorbing near 741 nm and fluorescing weakly near 760 nm at 77 K. Adsorption isotherms indicate some degree of cooperativity in the binding of Chl and DMMA to the particles. The photoreduction of p-dinitrobenzene by hvdrazobenzene. scnsitized by these particles, has been demonstrated     

ACTION SPECTRA OF CATION EFFECTS ON THE FLUORESCENCE POLARIZATION AND INTENSITY IN THYLAKOIDS AT ROOM TEMPERATURE

Abstract— The degree of polarization of chlorophyll- a (Chl- a ) fluorescence is known to monitor the extent of excitation migration and/or the orientation of the photosynthetic pigment molecules. We report here the effects of cations, at room temperature, on the degree of polarization of Chl- a fluorescence, and fluorescence intensity in thylakoids as a function of excitation wavelength. Observations of maxima at 650 and 675 nm in the cation-induced changes in the excitation spectrum for fluorescence at 730 and 762 nm, and, in the action spectra for the depolarization of fluorescence lead us to suggest that the regulation of the initial distribution of excitation to photosystem II involves the better coupling of Chl- b and- a in the light harvesting complex with Chl- a in the reaction center II complex.     

Electron paramagnetic resonance, optical absorption and IR spectroscopic studies of the sulphate mineral apjohnite

Apjohnite, a naturally occurring Mn-bearing pseudo-alum from Terlano, Bolzano, Italy, has been characterized by EPR, optical, IR and Raman spectroscopy. The optical spectrum exhibits a number of electronic bands around 400 nm due to Mn(II) ion in apjohnite. From EPR studies, the parameters derived, g=2.0 and A=8.82 mT, confirm MnO(H(2)O)(5) distorted octahedra. The presence of iron impurity in the mineral is reflected by a broad band centered around 8400 cm(-1) in the NIR spectrum. A complex band profile appears strongly both in IR and Raman spectra with four component bands around 1100 cm(-1) due to the reduction of symmetry for sulphate ion in the mineral. A strong pair of IR bands at 1681 and 1619 cm(-1) with variable intensity is a proof for the presence of water in two states in the structure of apjohnite.     

PHOTOPHYSICAL PROPERTIES OF PORPHYRIN-CHLORIN SYSTEMS IN THE PRESENCE OF SURFACTANTS

Abstract The therapeutic efficacy of PDT is related to the capability of the photosensitizer to absorb light at a wavelength that can penetrate into tissues. We have synthesized two systems, a haematoporphyrin-chlorin (HPC) and a dihaematoporphyrin ether or ester (DHE) with the terminal ring converted to a chlorin (DHEC). The presence of the chlorin moiety provides an extra band at ˜ 660 nm with a relative amplitude from 5 to 10 times larger than that of the porphyrin at 630 nm. Since both HPC and DHEC strongly aggregate in buffer, we have studied their photophysical properties in the presence of cationic surfactants at different concentrations below and above the critical micelle concentrations. Absorption spectra were measured together with emission spectra and fluorescence decays at different observation wavelengths under excitation at 364 nm. The results were compared with those obtained for DHE in the same environmental conditions. As for DHE, the presence of micelles disaggregate both compounds, resulting in a large increase in the relative emission intensity at ˜ 670 nm due to the presence of the chlorin moiety. The fluorescence decays could be fitted by two or three exponential components indicating the presence of more than one molecular species and/ or conformations. On the basis of our measurements the chlorin molecule does not seem to modify appreciably the photophysical properties of the porphyrin molecules but does superimpose its absorption and emission spectrum onto that of the porphyrin. This result may be of relevance in the possible use of these compounds in PDT.     

SPECTROSCOPIC CHARACTERIZATION OF TWO FORMS OF THE D1-D2-CYTOCHROME b559 COMPLEX FROM SUGAR BEET

Two D1-D2-cytochrome b559 complex forms, called RCIIa and RCIIb, with different pigment stoichi-ometry were characterized using absorption and surface-enhanced resonance Raman scattering spectroscopy and spectral gaussian deconvolution. Electronic absorption spectra of the RCIIb at 277 K showed significant differences compared to RCIIa, i.e . a strong decrease in the absorbance due to carotenoid and chlorophyll for the same amount of pheophytin. A reduced carotenoid and chlorophyll content in RCIIb was also observed in the surface-enhanced resonance Raman scattering spectra. Spectral deconvolution elicited three main absorption bands at 680, 672 and 669–670 nm, which were ascribed to P680, pheophytin and accessory chlorophyll, respectively. In addition, a minor component around 667 nm was observed in the RCIIb, most probably due to some reaction center inactivation. Calculation of the relative area under the gaussians together with pigment stoichiometry data suggest that the 680, 672 and 669–670 nm components contain, respectively, two chlorophylls, two pheophytins and four chlorophylls for the RCIIa, and two chlorophylls, two pheophytins and two chlorophylls for the RCIIb.     

Laser action and excited state absorption of chlorophyll-a

Stimulated emission of chlorophyll-a is observed in the spectral range around 670 nm. The calculated wavelength at the threshold turns out to be shifted by 20 nm towards longer wavelengths. To clarify this discrepancy, extrinsic-loss variations at the laser as well as measurements of nonlinear are carried out. The results indicate an excited singlet state absorption (σ > 10¹⁷ cm² within the range from 670 to 700 nm).     

FLUOROMETRIC ANALYSIS OF THE LONG-WAVELENGTH ABSORPTION BAND OF N-7 METHYLATED GMP INTO THE CONSTITUENT BANDS OF THE TWO ELECTRONIC STATES

Steady-state measurements of fluorescence anisotropy are used to resolve the long-wavelength absorption spectrum of 7-methyl guanosine 5'-monophosphate (GMP) in pH 5 buffer at room temperature into component spectra that correspond to the electronic transitions I and II present in that spectral region. We have chosen this derivative of guanine because its fluorescence quantum yield is much greater than that of GMP. It is found that the data are adequately described by a model that involves emission exclusively from state I, with state II converting to it with 100% efficiency. The shape of the absorption spectrum of state II is virtually independent of the angle theta between the absorption transition dipole moments of states I and II, whereas that of state I is dependent on theta. We analyze the data on the basis of the premise that in the short-wavelength region state II is the predominantly absorbing state. This premise is based on studies of single-crystal polarized reflection and linear dichroism from stretched films. The spectral maxima for the two states are found to be at about 290 and 260 nm, respectively. There is also a weak band which is centered at about 245 nm. The oscillator strengths are found to be 0.07, 0.21 and approximately 0.04, for states I, II and that associated with the weak band, respectively. The importance of these findings with regard to the photophysical properties of nucleic acids and calculations of their CD spectra is discussed.