PHOTOREACTIONS OF THE CHROMOPHORE CATIONS OF DENATURED SPECIES OF C-PHYCOCYANIN AND ALLOPHYCOCYANIN

Abstract— C-phycocyanin dissolved in buffer containing 75% ethylene glycol (vol/vol) shows photorevers-ible reactions which are ascribed to the interconversion by light of two species: PC_r, PC_g. After denaturation with formic acid, the chromophore cation of PC_g, can be irreversibly photoconverted to that of PC_r A conversion of the chromophore of denatured PC_g, to that of denatured PC_r also occurs after adjustment of the pH to about 8. Under the same conditions the chromophore of allophycocyanin shows similar reactions. Moreover, the reactions of the chromophores of the denatured species of these phycobiliproteins are similar to the reactions of the chromophore of the denatured species P_fr of photochrome.     

A POSSIBLE CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTOR OF CHLOROPHYLL SYNTHESIS IN THE GREEN ALGA Ulva rigida

Chlorophyll synthesis is stimulated by red light pulses in the green alga Ulva rigida C. Aghard. Chlorophyll synthesis in darkness is greater after longer red light pulses (30 min) than after shorter red light pulses (5 min). Chlorophyll synthesis was higher after red light pulses of 14 Wm_-2 fluence rate than after those of 7 Wm_-2. The effect of red light showed some far-red reversibility. The reversion by far-red light was higher after red light pulses of 4 min than after those of 30 min. These results indicate the existence of a rapid induction of chlorophyll synthesis during the red light pulses and a fast escape from photoreversibility. The percentage of reversion is also affected by the fluence rate of the light pulses. The reversion was reduced by about 15% when the photon fluence rate was increased from 7 to 14 Wm_-2. Reversion was also observed when red and far-red light pulses were applied successively. Thus, phytochrome or a phytochrome-like photoreceptor could be involved in the induction of chlorophyll synthesis in Ulva rigida.     

THE FORMATION OF TWO FORMS OF BATHORHODOPSIN AND THEIR OPTICAL PROPERTIES

Abstract— Using two kinds of rhodopsin preparations (digitonin extract and rod outer segments suspension), we measured changes in absorption spectra during the conversion of rhodopsin or isorhodopsin to a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (437 nm) at 77 K and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (> 650 nm) at 77 K. The reaction kinetics could be expressed with only one exponential in the former case and with two exponentials in the latter case. These data suggest that both rhodopsin and isorhodopsin are composed of a single molecular species, while bathorhodopsin is composed of two molecular species, designated as bathorhodopsin₁ and bathorhodopsin₂. The absorption spectra of these bathorhodopsin were calculated by two different methods (kinetic method and warming-cooling method). The former was based on the kinetics of the conversion of two forms of bathorhodopsin by irradiation with the red light. The spectra obtained by this method were consistent with those obtained by the warming-cooling method. Bathorhodopsin₁ and bathorhodopsin₂ have Λ_max at 555 and 538 nm, respectively. The two forms of bathorhodopsin are interconvertible in the light, but not in the dark. Thus, we suggest that a rhodopsin molecule in the excited state relaxes to either bathorhodopsin₁ or bathorhodopsin₂ through one of the two parallel pathways.     

EVIDENCE FOR A SEQUENTIAL PATHWAY FROM Pr TO Pfr OF THE PHOTOTRANSFORMATION OF 124-kDa OAT PHYTOCHROME

Abstract. Phototransformation kinetics of 124-kDa oat phytochrome at 298 K after a red (660-nm) laser flash excitation were recorded at different wavelengths. The kinetics of the dark relaxation processes for lumi-R to P_fr can be satisfactorily described by only 3 rate constants: k = 28000 s^-1 370 s^-1 and 20 s^-1. The first rate constant is due to the decay of lumi-R to meta -Ra. The latter two rate constants correspond to processes establishing the far-red (>700 nm) absorption band. No meta -Rb could be detected. From the wavelength dependency of the amplitudes of these two rates, parallel pathways in the formation of P_fr could be excluded. A unique sequential pathway for the dark relaxation leading to P_fr seems to be an intrinsic property of 124-kDa phytochrome, however. Assuming a sequential pathway, molar extinction coefficients for intermediates have been calculated. These values agree with molar extinction coefficients obtained from low-temperature spectra. The process with a rate constant of 370 s^-1 corresponds to absorbance changes for the formation of meta -Rc from meta -Ra and the rate constant of 20 s^-1 describes the absorbance changes due to the transformation of meta -Rc to P_fr.     

Recombinant Phytochrome of the Moss Ceratodon purpureus: Heterologous Expression and Kinetic Analysis of Pr→ Pfr Conversion

Abstract— The phytochrome-encoding gene Cerpu;PHY;2 ( CP2 ) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P φ B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [P_r]) far-red-absorbing phytochrome [P_fr]) for the PCB adduct, and 659/724 nm for the PφB-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that PφB is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophyto-chrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the P_r→ P_fr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I₇₀o)> decaying with a time constant of - 110 u.s. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the P_fr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.     

BIOLUMINESCENCE OF THE BRITTLE STAR OPHIOPSILA CALIFORNICA

The light-emitting principle of the brittle star Ophiopsila californica has been isolated and purified. It was found to be a green-fluorescent photoprotein (molecular weight 45000) which emits green light (λ_max 500 nm) when H₂O₂ is added, independently of the presence or absence of O₂. The green fluorescence (emission maximum 500 nm, excitation maximum 440 nm) spectrally coincided with the H₂O₂-triggered luminescence, indicating that the green fluorescent chromophore is the light-emitter of the photoprotein luminescence.     

PHYTOCHROME VARIATION AND REVERSION IN LEMNA MINOR, L. GIBBA G3 AND L. PAUCICOSTATA 6746

Abstract— Dual wavelength phot00ometry showed an increase of phytochrome in darkness in partially etiolated plants of three heterotrophically cultured Lemna species. In all three species, P_fr formed by a brief illumination decreased in subsequent darkness with half-lives of 2–6 h. In L. minor and L. gibba , dark reversion was observed during the first 8h of darkness; in L. paucicostata , however, only when the plants were in a slightly anaerobic condition. Under continuous illumination, phytochrome decay was not influenced by light intensity. Actinic doses of far red resulted in non-photoreversible changes of absorbance. In material exposed to red for longer than 12 h, these changes increased after preceding actinic doses of red light. This effect may result in erroneous phytochrome estimations.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

THE 518-mμ ABSORBANCE CHANGE AND ITS RELATION TO THE PHOTOSYNTHETIC PROCESS

Abstract— A study of the 518-mμ light-induced absorbance change in green cells and a comparison with photosynthetic O₂ evolution were made. The effect of various chemical agents was also investigated. On the one hand, no antagonistic two-light effect was observed on the absorbance change, and DCMU had only a partial inhibitory effect on it. On the other hand, it was possible to observe in some cases an indirect kinetic relationship between O₂ evolution and 518-mμ change. It is suggested that probably at least two substances absorb around 518-mμ, one ( X _I) belonging to system I and another ( X _II) to system II. The existence and the function of X _I are hypothetical and the identity of X _II with the first photoproduct of system II is yet not well established.     

THE AQUEOUS PHOTOLYSIS OF ETHYLENE GLYCOL ADSORBED ON GOETHITE

Abstract— Suspensions of goethite (α-FeOOH) were photolyzed in aerated ethylene glycol-water solutions at pH 6.5, with ultraviolet light in the wavelength range300–400 nm. Under these conditions, formaldehyde and glycolaldehyde were detected as photoproducts. Quantum yields of formaldehyde production ranged from 1.9 7times; 10^-5 to 2.9 × 10^-4 over the ethylene glycol concentration range of 0.002-2.0 mol/ℓ, and gave evidence that the reaction occurred at the goethite surface. Quantum yields of glycolaldehyde were 20% less than those of formaldehyde, and displayed a concentration-dependent relationship with ethylene glycol similar to that of formaldehyde. Immediately after photolysis, Fe²⁺ was measured to be 4.6 × 10^-7 mol/ℓ in an aerated suspension containing 1.3 mol/ℓ ethylene glycol, and 8.5 × 10^-6 mol/ℓ in the corresponding deoxygenated suspension. Glycolaldehyde was not generated in the deoxygenated suspensions. These results are consistent with a mechanism involving the transfer of an electron from an adsorbed ethylene glycol molecule to an excited state of Fe³⁺ (Iron[III]) in the goethite lattice, to produce Fe²⁺ and an organic cation. In a series of reactions involving O₂, FeOOH, and Fe²⁺, the organic cation decomposes to form formaldehyde and the intermediate radicals "OH and" CH₂OH. OH reacts further with ethylene glycol in the presence of O₂ to yield glycolaldehyde. Aqueous photolysis of ethylene glycol sorbed onto goethite is typical of reactions that can occur in the aquatic environment.     

MODE OF COACTION OF PHYTOCHROME AND BLUE LIGHT PHOTORECEPTOR IN CONTROL OF HYPOCOTYL ELONGATION

Abstract The rate of hypocotyl longitudinal growth in seedlings of Sesamum indicum L. is strongly inhibited by continuous blue light (cBL)† and slightly by continuous far-red light while continuous red light (cRL) or red light pulses are hardly effective from 60 h after sowing onwards. Between 36 and 60 h after sowing the growth rate responds to red light pulses the effect of which is fully reversible by long wavelength far-red light. When seedlings are kept in cBL for 3 days and then treated with red light hypocotyl growth rate responds strongly. However, RL effectiveness decreases with time after transfer from BL to RL. BL → darkness transfer experiments with different levels of P_fr established at the beginning of darkness show that after a BL pretreatment phytochrome (P_fr) alone is capable of fully controlling growth rate. When white light (WL) is given no BL effect is detectable in weak WL. Only high light fluxes maintain a typical BL growth rate. At medium WL fluxes elongation rate returns gradually to the dark rate. The simplest explanation of the data is that light absorbed by a separate BL photoreceptor is necessary to maintain responsivity to P_fr. With increasing age of the seedlings the requirement for BL increases strongly. On the other hand, brief light pulses—given to demonstrate photoreversibility of phytochrome—remain equally effective provided that responsivity to P_fr exists.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

BIOPHYSICAL AND BIOCHEMICAL ASPECTS OF PHENGODID (RAILROAD-WORM) BIOLUMINESCENCE

In studies of the bioluminescence of 11 species of phengodid collected in central and southeast Brazil, we have found that: (1) their lateral lanterns emit light in the yellow-green region (λ_max= 540–580 nm) and the head lantern color is shifted to the red region ( λ_max= 565–620 nm), (2) the luciferins of both types of lanterns are identical to that of lampyrids and elaterids and (3) the luciferase physicochemical properties are also similar to those of lampyrids and elaterids (optimum pH ca 8.1; K_m(ATP) = 260–370 μM , Kμ(luciferin) = 170–400 μM; molecular weight ca 60 kDa; apparent activation energy of in vitro bioluminescence ca 58 kJ/mol). Thus the bioluminescence system of phengodids appears to be essentially the same as that of lampyrids and elaterids. The different bioluminescence colors of the lanterns of Phrixothrix species (λ_head= 600–620 nm; λ_lateral= 535–565 nm) and other phengodid species are probably elicited by the presence of luciferase isoenzymes, as occurs in the case of elaterid prothoracic and abdominal lanterns.     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

CHEMILUMINESCENCE IN THE PEROXIDATION OF TANNIC ACID

Abstract— Peroxidation of tannins with alkaline H₂O₂ is accompanied by weak chemiluminescence in the spectral region 480–800 nm. o-Di and tri-hydroxy groups of polyphenols undergo oxidation by a free-radical mechanism and a green intermediate anion-radical with absorption Δ_max= 600 nm is formed. The radical mechanism is supported by the low activation energy 14–20 kJ/mol and the quenching effect of radical scavengers. The reaction of the green intermediate with peroxy anions is the chemiluminescence rate limiting step. In the presence of a-hydroxy-methylperoxide formed from H₂O₂ and formaldehyde, the alkaline peroxidation of tannins is accompanied by strong red luminescence (420–800 nm). The base catalyzed decomposition of peroxides gives only a weak red emission (460–800 nm). Light intensity is enhanced in D₂O by a factor 6.5. Quenchers of O₂(¹Δ_g) and 1,3-di-phenylisobenzofurane diminish light intensity in non-aqueous solutions. The data suggest ¹O₂ participation in the observed chemiluminescence. Thermo-chemical calculations give —ΔH values from 250–1000 kJ/mol for one elementary reaction step which limits the mechanism of chemi-enereization. Chemiexcitation of tannins is relevant to biochemical mechanisms of aerobic degradation of aromatic compounds, energy utilization as well as to defense and resistance processes in plants.     

PHYTOCHROME INTERMEDIATES IN VIVO– III KINETIC ANALYSIS OF INTERMEDIATE REACTIONS AT LOW TEMPERATURE

Abstract— Kinetic experiments have provided evidence for a series of light and dark reactions of phytochrome intermediates at low temperature in Pisum epicotyl tissue. A photoequilibrium exists between P_r and P₆₉₈, and between P_fr and P₆₅₀. A dark reversion of P₆₉₈P_r and P₆₅₀p_fr at –70°C has been demonstrated. When cooled to 70°C under incandescent light, most of the phytochrome in the tissue is driven into photochemically unreactive intermediates. About 2% of the phytochrome remains as weakly absorbing intermediates that form P_r and P_fr in darkness. A scheme is presented for phytochrome phototransformation in vivo.     

Distinctive Properties of Dark Reversion Kinetics between Two Red/Green‐Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis

Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP‐phosphodiesterase/adenylate cyclase/FhlA (GAF) domain and exhibit reversible photoconversion. Red/green‐type CBCR GAF domains that photoconvert between red‐ (Pr) and green‐absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green‐type CBCR GAF domains. We previously reported that AnPixJ's second domain (AnPixJg2) but not its fourth domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr‐to‐Pg photoconversion and rapid Pg‐to‐Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site‐directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300‐fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF domains with adenylate cyclase catalytic regions, such as AnPixJg2‐AC, AnPixJg4‐AC and AnPixJg2_DR6‐AC. We detected successful enzymatic activation under red light for both AnPixJg2‐AC and AnPixJg2_DR6‐AC, and repression under green light for AnPixJg2‐AC and under dark incubation for AnPixJg2_DR6‐AC. These results provide platforms to develop cAMP synthetic optogenetic tools.     

LIGHT-INDUCED LINEAR DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS–II. CHROMOPHORE ROTATION IN IMMOBILIZED PHYTOCHROME

-Large phytochrome immobilized via anti-phytochrome immunoglobulin bound to Sepharose beads was irradiated to saturation with unpolarized far-red light. The apparent absorbance level was recorded in a dual wavelength spectrophotometer with both measuring beams set to either 660 or 730 nm and polarized perpendicular to each other. The sample was then irradiated with red polarized light. The apparent change in absorbance obtained after this irradiation indicated that purified phytochrome could show linear dichroism. From the absorbance values obtained it was computed that the direction of the long-wavelength transition moment changes by either 32 or 148^o, when phytochrome is transformed from P_r to P_fr. Considering the model of Hahn and Song (1981) the latter value appears more likely. In light of these results, the conclusions drawn from in vivo experiments on action dichroism in Dryopteris (Etzold, 1965), Adiantum (Kadota et al., 1982) and Mougeoutia (Haupt. 1970), which point to a 90^o rotation. should be reconsidered.     

Intraperitoneal Photodynamic Therapy: Comparison of Red and Green Light Distribution and Toxicity

Abstract— The aim of this study was to compare red (652 nm) and green (514 nm) light for photodynamic therapy (PDT) of the peritoneal cavity with emphasis on light distribution and toxicity. Red-light PDT was limited by intestinal toxicity and it was hypothesized that less penetrating green light would allow higher light doses to be used in the peritoneal cavity. Female non-tumor-bearing rats were photosensitized with mTHPC (meta-tetrahydroxyphenylchlorin, Foscan®) intravenously or intraperitoneally and the peritoneum was illuminated using a minimally invasive technique. For both red and green light, the time of illumination was varied to give the required dose. Light fluence rate was measured in situ at multiple sites within the abdominal cavity. The toxicity experiments were carried out with a total of 160 J incident red or 640 J incident green light and a drug dose of 0.15 mg/kg Foscan® For red light a mean fluence rate of 55.2 38.5 mW cm ₂ was measured, with a peak fluence rate of 128 mW cm ₂ on the intestines. For green light the mean and peak fluence rates were 8.2 9.0 (i.e. including zero fluence rate measurements) and 28 mW cm ₂, respectively. Intestines were most vulnerable to red light illumination. The intravenous injection route resulted in increased toxicity for red light, but for green light there were no major differences between intravenous and intraperitoneal routes. The 4 h interval between drug and illumination resulted in very little toxicity for both wavelengths. We conclude that for intraperitoneal PDT green light allows higher light doses than red light, but the light distribution over the peritoneum is much less favorable and may not be suitable for whole peritoneal illumination using a minimal-access technique.     

ENERGY TRANSFER IN THE ACCESSORY PIGMENTS R-PHYCOERYTHRIN AND C-PHYCOCYANIN

Abstract— The photosynthetic accessory pigments R-phycoerythrin and C-phycocyanin were extracted from a red sea-weed and a filamentous blue-green alga. Investigation of quenching of phycoerythrin fluorescence by phycocyanin at room and liquid nitrogen temperatures yielded rates for the SternVolmer quenching constants ( K_q From these values, rate constants k_(D–A) were calculated for the transfer of energy from phycoerythrin to phycocyanin.
The transfer rate constants obtained were such that a collisional transfer mechanism was ruled out, and were of similar order of magnitude as the rate constants for transfer of electronic energy in mixed organic crystals.