Light-induced absorbance changes in cell-free extracts of Neurospora crassa.

Abstract— The mycelium of Neurospora crassa was ground and extracted with buffer and separated into a soluble supernatant fraction and a particulate fraction by centrifugation. Both fractions were examined for light-induced absorbance changes. Irradiation of the supernatant fraction caused a reversible photooxidation of cytochrome c which was inhibited by sodium azide or by dialysis. The action spectrum for the photooxidation showed that the response was mediated by an endogenous flavin. The photooxidation of cytochrome c, lost by dialysis, could be restored by adding flavin mononucleotide. Irradiation of the particulate fraction with blue light caused a reversible photoreduction of cytochrome c and cytochrome oxidase and, in some samples, of cytochrome b as well. The supernatant fraction showed photooxidation of cytochrome c rather than the more usual photoreductive changes because of the presence of super-oxide dismutase activity.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.     

ACTION SPECTRA FOR THE ABSORBANCE CHANGE AT 880 nm AND FOR P870 FLUORESCENCE FROM A PHOTOSYNTHETIC REACTION CENTER*

Abstract— Photosynthetic reaction centers isolated from blue-green mutant strain R-26 of Rhodopseudomonas spheroides were used to study the action spectra for the light-induced absorption changes at 880 nm, and for the P870 fluorescence. These spectra indicated that a pigment, possibly bacteriopheophytin, with an absorption maximum at 757 nm is an intrinsic component of the reaction center and transfers energy to P870 with relatively high efficiency.     

LIGHT-INDUCED ABSORBANCE CHANGES IN CHLOROPLASTS AT -196°C*

Abstract— Absorbance changes induced by irradiating chloroplasts at — 196°C were measured in the region of 525–575 nm with a single-beam spectrophotometer. Irradiation at low temperature caused a bleaching at 556 nm due to oxidation of cytochrome b₅₅₉ but little or no change of cytochrome f. There occurred in addition a loss of absorbance at 547 nm and an increase at 543 nm. The bleaching at 547 nrn (and possibly the increase at 543 nm) could be induced chemically with dithionite or borohydride but not ascorbate. Subchloroplast particles with only Photosystem I activity showed no light-induced absorbance changes, while particles containing combined Photosystem I and Photosystem II activities showed the same changes as whole chloroplasts. Scenedesmus mutant No. 11 cells showed no absorbance changes while mutant No. 8 and wild-type cells showed the normal changes. It is concluded that the photooxidation of cytochrome b₅₅₉ and the photoreduction causing the bleaching at 547 nm are both mediated by Photosystem II.     

STUDIES ON THE RED AND BLUE FORMS OF THE PIGMENT OF BLEPHARISMA*

Abstract— –The intracellular pigment of the ciliate protozoan Blepharisma in the presence of oxygen sensitizes the cells to bright visible light (2700 foot candles (fc)). Illumination of the cells with dim visible light (200 fc) changes the color of the pigment to blue-gray; such cells are no longer sensitive to bright visible light. The pigment which exists in granules can be extruded by cold treatment and is slowly regenerated. The suspension of red cells, the extruded pigment from them and an ethanol extract of the pigment all have very similar absorption spectra. Illumination of the red pigment in ethanol converts it to the blue form of the pigment but only if oxygen is present, indicating a photooxidation. The pigment can be oxidized in darkness to the blue form by ozonation. A suspension of blue cells, the extruded pigment from them and an ethanol extract from them, all have similar absorption spectra. The pigments in red and blue form are very similar spectrophotometrically and in solubility in three species of Blepharisma studies: B. americanum, B. intermedium and B. japonicum. The purified pigment has strong absorption in the far (200–300 nm) ultraviolet (u.v.) and may serve as a screen against damaging U.V. radiation, especially as Blepharisma shows poor photoreactivation.     

PHOTOTACTIC ORIENTATION BY THE CILIATE, STENTOR COERULEUS

Stentor coeruleus exhibits negative phototaxis to visible light, in addition to a step-up photophobic response. The negative phototaxis was established by demonstrating the swimming of Stentor toward a focused beam away from the light source. The action spectrum showed a maximum at 610–620 nm and is essentially identical to that of the step-up photophobic response. Proton uncouplers such as micromolar concentrations of FCCP and TPMP⁺ inhibited the negative phototaxis.     

SENSITIZATION OF PHOTOREACTIONS IN EIMHJELLEN'S RHODOPSEUDOMONAS BY A PIGMENT ABSORBING AT 830 mμ*

Abstract— The excitation spectrum for bacteriochlorophyll b fluorescence, the action spectrum for cytochrome-553 oxidation, and the action spectrum for P-985 bleaching are compared to the absorption (1-T) spectrum of a Rhodopseudomonas sp. NHTC 133 extract over the range 770 to 930 mμ. These spectra show that a minor pigment P-830 is more effective in sensitizing cytochrome oxidation and P-985 bleaching than in exciting fluorescence of Bchl b. These results are consistent with the proposal that P-830 is a form of Bchl b in special relation to the reaction center pigment P-985.     

NEW SPECTRAL COMPONENTS IN HIGH RESOLUTION ABSORPTION SPECTRA OF GREEN BACTERIAL REACTION CENTER COMPLEXES AT 5K*

Abstract—absorption spcctra of reaction center Complexes I and II from Chlorobium limicola f. thiosul-fatophilum were taken from 760 and 860 nm at 5 K. Fourth and eighth derivatives of the spectra were calculated from the digital data. Light-minus-dark difference spectra were taken, also at 5 K, with 590 nm actinic light. A shoulder not visible at 77 K appears on the long wavelength side of the 834 nm peak in Complex I. In Complex II, which is derived by guanidine HCI treatment of I, the shoulder is much more pronounced; derivative peaks appear at 834 and 838 nm. In the difference spectra, there are troughs at 832 and 838 nm. The latter trough is the first instance in green bacteria of a wavelength coincidence between a light-induced bleaching and a peak in (derivative) absorbance. There is also a nearly symmetrical pair of features, a trough at 814 nm and a peak at 818 nm, that appear to represent a light-induced bathochromic shift of the absorbance at 816 nm, a peak which occurs in both complexes as well as the photochemically inert bacteriochlorophyll a (Bchl a) protein. Other features in the absorption spcctra of both Complexes occur at virtually the same wavelengths as the peaks in purified Bchl a-protein trimer. We conclude that a large fraction of the Bchl a in Complex II is in a conformation similar to that of a single subunit of Bchl a-protein.     

Multichannel flash spectroscopy of the reaction centers of wild-type and mutant Rhodobacter sphaeroides: bacteriochlorophyllB-mediated interaction between the carotenoid triplet and the special pair

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light-induced spectral changes in the 350-1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870(+)QA(-), and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870(+)) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P870(+), and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 micros). The bleaching at 800 nm arises from the pigment interaction between P870(+) and the accessory bacteriochlorophylls on A and B branches (BA,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals--a sharp peak at 809 +/- 2 nm and a broad band at 870 nm--which decay at the same rate as the TmS spectrum of Car (ca 10 micros). The former is ascribed to the perturbation, by Car(+), of the absorption spectrum of BB; the latter, to the TmS spectrum of P870(+), a species that appears to be in approximate thermal equilibrium with Car(+). These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.     

Biphasic fluence-response curves and derived action spectra for light-induced absorbance changes in Phycomyces mycelium

The photoresponses of Phycomyces, including phototropism and photocontrol of sporangiophore development, are mediated primarily by blue and UV light. Recent results on these two responses indicated a subsidiary role for green light. We have measured in vivo light-induced absorbance changes (LIAC) in mycelial samples of a caroteneless (carB) strain to compare the effectiveness of UV, blue, and green light. In the dual-wavelength kinetic mode of the spectrophotometer, measuring wavelengths of 445 and 470 nm were chosen, because green light produces substantial absorbance changes between these two wavelengths. Fluence-response curves were measured for 13 wavelengths between 365 and 530 nm, and for variable exposure times between 0.5 and 320 s. With one exception (365 nm), the curves were biphasic. The low fluence component was generally sigmoidal with an abrupt rise. The high fluence component failed to reach saturation for the fluences tested (less than 70 μmol m⁻² s⁻¹). Using the inferred threshold fluences of these two components as criterion effects, we obtained two action spectra. For the low fluence component, the action spectrum showed major peaks at 394, 450, and 530 nm and a minor peak at 416 nm. The high fluence component action spectrum showed very little sensitivity in the blue region. The major sensitivity was in the near UV, and a relatively small peak appeared in the green part of the spectrum at 507 nm. The biphasic character of the fluence-response curves suggests that two photosystems are responsible for the absorbance changes. The low fluence photosystem is sensitive mainly to blue and UV light and may thus represent a physiological blue-light photoreceptor. The high fluence photosystem is clearly not of this type. It (and perhaps the low fluence system as well) may mediate some of the subsidiary physiological effects of green light.     

The Photoreceptor for Phototaxis in the Photosynthetic Flagellate Euglena gracilis

The unicellular flagellate Euglena gracilis shows positive phototaxis at low fluence rates (≤10 W m ²) and negative phototaxis at high fluence rates (≥100 W m ²). Currently, retinal or flavins/pterins are discussed as chromo-phores of the photoreceptor. When grown in the presence of 4 mM nicotine, a retinal inhibitor, for several generations, the cells still showed both responses, indicating that retinal is unlikely to be the chromophoric group of the photoreceptor responsible for phototaxis. The native flavin(s) can be substituted by growing the cells in roseo-flavin dissolved in the medium. The absorption spectrum of roseoflavin extends well beyond the action spectrum for phototaxis (up to 600 nm). Excitation at wavelengths >550 nm does not cause phototactic orientation in control cells but causes both positive and negative phototaxis in roseoflavin-grown cells, indicating an uptake and assembly of the chromophore in the photoreceptor complex. The white mutant strain 1224-5/1f, induced by streptomycin treatment, lacks flavins as indicated by fluorescence spectroscopy. The phototaxis-deficient pheno-type cannot be complemented by the addition of external riboflavin. Fluorescence spectra of intact paraxonemal bodies (PAB) indicate that both pterins and flavins are involved in photoperception and that the excitation energy is efficiently funneled from the pterins to the flavins. This energy transfer is disrupted by solubilization of the PAB. In intact PAB flavins are not accessible to reducing or oxidizing substances, indicating that they are located inside the structure, while pterins are accessible, so that their localization can be assumed to be on the surface. The results described above are discussed with regard to the potential involvement of flavins and pterins as well as retinal in photoperception.     

BIOCHEMICAL ISOLATION AND SPECTROSCOPIC CHARACTERIZATION OF POSSIBLE PHOTORECEPTOR PIGMENTS FOR PHOTOTAXIS IN A FRESHWATER Peridinium

Photoreceptor pigments have been isolated biochemically from the freshwater dinoflagellate Peridinium gatunense, and characterized spectroscopically. At least four different chromoproteins can be detected in the crude extract and the membrane fraction isolated from the cells absorbing at 580, 638, 667 and 710 nm, which correspond with the maxima in the action spectrum for phototaxis in this organism. Light energy absorbed by shorter wavelength pigments is emitted as fluorescence at wavelengths which are absorbed by pigments with maxima at longer wavelengths. Protein separation on a MonoQ anion exchanger column using fast liquid chromatography resulted in a non-bound fraction and four bound fractions eluted by an NaCI gradient, which differed in their pigment composition.     

Photomechanical transduction in Amoeba proteus: An action spectrum

An action spectrum for the light-induced stop reaction of amoeboid movement in Amoeba proteus has been determined. The amoebae show a sensitivity in the blue with a peak between 440 and 470 nm. This agrees with the older qualitative observations (M. R. Harrington and E. Leaming, Am. J. Physiol., 3 (1900) 9 - 16; S. O. Mast, J. Exp. Zool., 9 (1910) 265 - 277) but no positive phototaxis was found (A. A. Schaeffer, Biol. Bull., 32 (1917) 45 - 74). Absorption spectra of suspensions of whole cells have an absorption band with a similar spectral peak. The response follows the Bunsen-Roscoe law and the sensitivity appears to be greater at low oxygen tension. The photomechanical link in this response, i.e. conversion of a light stimulus to a change in motion, suggests a primitive mechanism of avoidance that may have evolutionary implications (R. M. Eakin, Evol. Biol., 2 (1968) 194 - 242; L. von Salvini-Plawen and E. Mayr, Evol. Biol., 10 (1977) 207 - 263).     

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

Absorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light-induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light-induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

ON THE PHOTORECEPTOR PIGMENT FOR PHOTOTROPISM AND PHOTOTAXIS: IS A CAROTENOID THE MOST LIKELY CANDIDATE?*

Abstract— The absorption spectrum of lycopene can be altered to show significant absorption in the 350–360 nm region in an ethanol-water mixture, thus resembling the phototropic action spectrum of Avena coleoptiles. The hypochromicity (bleaching) of the main absorption band and appearance of the new band at 350–360 nm can be attributed to exciton interactions between two stacked lycopene molecules. β-Carotene does not show anomalous bleaching under identical conditions. Thus, the apparent modification of the absorption spectra of carotenoids in ethanol-water mixtures cannot be used as an argument to resolve the action spectrum in terms of carotenoids. In addition, we have critically reviewed the spectroscopic characteristics of carotenoids. Short lifetimes of the excited singlet states and inefficient intersystem crossing of carotenoids are not compatible with the suggestion that carotenoids are the most likely candidate for the photoreceptor pigment in phototropism.     

Photoactivation mechanism of orthovanadate-like (V=O)O3 species

The photoluminescence spectrum and action spectrum for the photooxidation of orthovanadate-like (V=O)O₃ species exhibiting photoluminescence at 520 nm indicate that the triplet excited state T₁ of the orthovanadate-like species, which is formed from the singlet excited states S₁ and S₂ by intersystem crossing, is directly involved in the photooxidation of cyclohexane into cyclohexanone in the presence of molecular oxygen.     

Combinational Biosynthesis of a Fluorescent Cyanobacterial Holo-α-Phycocyanin in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Using One Expression Vector

The phycobiliproteins (PBSs) are large pigment proteins found in certain algae that play a central role in harvesting light energy for photosynthesis. Phycocyanin (PC) is one type of PBSs that gains increasing attention owing to its various biological and pharmacological properties. In this paper, an expression vector containing five essential genes in charge of biosynthesis of cyanobacterial C-phycocyanin (C-PC) holo-α subunit (holo-CpcA) was successfully constructed resulting in over-expression of a fluorescent holo-CpcA in E. coli BL21. The vector harbored two cassettes: one cassette carried genes hox1 and pcyA required for conversion of heme to phycocyanobilin (PCB), and the other cassette carried cpcA encoding CpcA along with cpcE and cpcF both of which were necessary and sufficient for the correct addition of PCB to CpcA. The vector system contained a His-tag for protein purification. The purified protein showed correct molecular weight on SDS-PAGE gel and emitted orange fluorescence by UV excitation. The maximum peak of absorbance spectrum was at 623 nm, and the maximum peak of fluorescence emission and excitation were at 648 and 633 nm, respectively, which were similar to those of native C-PC. This study provides an efficient method for large-scale production of the fluorescent holo-CpcA in biotechnological applications. Guan and Qin contributed equally to this study.     

Ligninases fromChrysonilia sitophila (TFB-27441 strain)

Ligninase found in the extracellular medium of cultures ofChrysonilia sitophila was purifieded by ion exchange chromatography. Sodium dodecylsulfate/polyacrylamide gel electrophoresis allowed the determination of 68,000, 48,300, and 48,000 daltons for the molecular weights of ligninase I, II, and III, respectively. The absorption spectrum of the enzymes indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme II at 400 nm decreased in the presence of one equivalent of hydrogen peroxide. With an additional equivalent of phenol the maximum at 400 nm shifted to 417 nm. This spectrum is similar to horseradish peroxidase compound II. The pyridine hemochromogen absorption spectrum and iron content indicated that ligninases I, II, and III contained a Fe/heme ratio values of 0.8, 1.3, and 1.2 by a molecule of protein, respectively. These enzymes oxidize lignin efficiently, followed by the fluorescence technique and by the photon emission method.     

PHOTOTAXIS IN DICTYOSTELIUM DISCOIDEUM AMOEBAE

Abstract— An apparatus has been developed to measure phototactic movement in a population of amoebae of Dictyostelium discoideum. Fluence–response curves in white light show a positive phototaxis to light below 100mW/m². Higher intensities cause a negative phototaxis. An action spectrum, based on the zero-crossing points in fluence–response curves for monochromatic light, shows a major peak at about 405nm and secondary maxima at about 450, 520, 580 and 640nm. This action spectrum resembles the action spectra for accumulations of amoebae in and dispersal from light traps and that of inhibition of aggregation by light, but is distinctly different from the action spectrum for phototaxis by D. discoideum pseudoplasmodia.