Light-induced Spectral Changes Research Articles

Difference FTIR Spectroscopy of Jumping Spider Rhodopsin-1 at 77K.

Animal visual rhodopsins can be classified into monostable and bistable rhodopsins, which are typically found in vertebrates and invertebrates, respectively. The former example is bovine rhodopsin (BovRh), whose structures and functions have been extensively studied. On the other hand, those of bistable rhodopsins are less known, despite their importance in optogenetics. Here, low-temperature Fourier-transform infrared (FTIR) spectroscopy was applied to jumping spider rhodopsin-1 (SpiRh1) at 77 K, and the obtained light-induced spectral changes were compared with those of squid rhodopsin (SquRh) and BovRh. Although chromophore distortion of the resting state monitored by HOOP vibrations is not distinctive between invertebrate and vertebrate rhodopsins, distortion of the all-trans chromophore after photoisomerization is unique for BovRh, and the distortion was localized at the center of the chromophore in SpiRh1 and SquRh. Highly conserved aspartate (D83 in BovRh) does not change the hydrogen-bonding environment in invertebrate rhodopsins. Thus, present FTIR analysis provides specific structural changes, leading to activation of invertebrate and vertebrate rhodopsins. On the other hand, the analysis of O-D stretching vibrations in D2O revealed unique features of protein-bound water molecules. Numbers of water bands in SpiRh1 and SquRh were less and more than those in BovRh. The X-ray crystal structure of SpiRh1 observed a bridged water molecule between the protonated Schiff base and its counterion (E194), but strongly hydrogen-bonded water molecules were never detected in SpiRh1, as well as SquRh and BovRh. Thus, absence of strongly hydrogen-bonded water molecules is substantial for animal rhodopsins, which is distinctive from microbial rhodopsins.

Dynamic interplays between three redox cofactors in a DNA photolyase revealed by spectral decomposition

DNA repair catalyzed by photolyases is accomplished by a light-dependent electron transfer event from a fully reduced flavin adenine dinucleotide to a DNA lesion site. Prokaryotic DNA photolyase, PhrB, possesses a ribolumazine cofactor and a four-iron-four-sulfur cluster in addition to the catalytic flavin, but their functional roles are poorly understood. Here, we employ time-resolved absorption spectroscopy to probe light-induced responses in both solution and single crystals of PhrB. We jointly analyze a large collection of light-induced difference spectra from the wild-type and mutant PhrB obtained under different light and redox conditions. By applying singular value decomposition to 159 time series, we dissect light-induced spectral changes and examine the dynamic interplay between three cofactors. Our findings suggest that these cofactors form an interdependent redox network to coordinate light-induced redox responses. We propose that the ribolumazine cofactor serves as a photoprotective pigment under intense light or prolonged illumination, while the iron-sulfur cluster acts as a transient electron cache to maintain balance between two otherwise independent photoreactions of the flavin and ribolumazine.

Spectrokinetic characterization of photoactive yellow protein films for integrated optical applications

In this paper, the photocycle of the dried photoactive yellow protein film has been investigated in different humidity environments, in order to characterize its nonlinear optical properties for possible integrated optical applications. The light-induced spectral changes of the protein films were monitored by an optical multichannel analyser set-up, while the accompanying refractive index changes were measured with the optical waveguide lightmode spectroscopy method. To determine the number and kinetics of spectral intermediates in the photocycle, the absorption kinetic data were analysed by singular value decomposition and multiexponential fitting methods, whose results were used in a subsequent step of fitting a photocycle model to the data. The absorption signals of the films were found to be in strong correlation with the measured light-induced refractive index changes, whose size and kinetics imply that photoactive yellow protein may be a good alternative for utilization as an active nonlinear optical material in future integrated optical applications.

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains, LOV/LOV Proteins

LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly(66) in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.

Exploring plant endomembrane dynamics using the photoconvertible protein Kaede

Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.

Light-induced Change of Configuration of the LHCII-Bound Xanthophyll (Tentatively Assigned to Violaxanthin): A Resonance Raman Study

Raman scattering spectra of light-harvesting complex LHCII isolated from spinach were recorded with an argon laser, tuned to excite the most red-absorbing LHCII-bound xanthophylls (514.5 nm). The intensity of the nu(4) band (at ca. 950 cm-1) corresponding to the out-of-plane wagging modes of the C-H groups in the resonance Raman spectra of carotenoids appears to be inversely dependent on the probing laser power density. This observation can be interpreted in terms of excitation-induced change of configuration of the protein-bound xanthophyll owing to the fact that the intensity of this particular band is diagnostic of a chromophore twisting resulting from its binding to the protein environment. The comparison of the shape of the nu(4) band of a xanthophyll involved in the light-induced spectral changes with the shape of the nu(4) band of the xanthophylls present in LHCII, reported in the literature, lets us conclude that, most probably, violaxanthin is a pigment that undergoes light-driven changes of molecular configuration but also the involvement of lutein may not be excluded. Possible physical mechanisms responsible for the configuration changes and physiological importance of the effect observed are discussed.

Single-organelle tracking by two-photon conversion

Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation--such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 degrees C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

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.

The light induced valence change of europium in Sr2SiO4 ? Eu involving transient crystal structureElectronic supplementary information (ESI) available: details of the experiment under different irradiation conditions, characterization by XRD and PL spectra of Ba2SiO4 ? Eu3+ and Ca2SiO4 ? Eu3+. See http://www.rsc.org/suppdata/jm/b4/b400515e/

Sr2SiO4 ∶ Eu3+ shows orange-red emission of Eu3+ substitutively present in two different Sr sites. The light-induced spectral changes from orange-red sharp line emission to yellow-white broad band are observed in Sr2SiO4 ∶ Eu at room temperature under irradiation with short UV or X-rays. The spectral changes are attributed to the optically assisted reduction of Eu3+ → Eu2+. The photoreduced Sr2SiO4 ∶ Eu shows emission containing contributions from both Eu2+ and Eu3+ in comparison to chemically reduced samples. This is explained on the basis of preferential reduction of Eu3+ present in Sr(1) sites under irradiation due to unsatisfied EuSr–O–Si bonds. The absence of photoactivity for Ba2SiO4 ∶ Eu3+ (space group = Pnam) as well as Ca2SiO4 ∶ Eu3+ (space group = P21/n) indicates that crystal structure plays an important role in the photoreduction of Sr2SiO4 ∶ Eu3+ because of the prevailing orientational as well as the positional disorder in the latter. Further, the orientationally disordered monoclinic random domains persist within the orthorhombic lattice of Sr2SiO4, resulting in the positionally disordered Sr atoms and orientationally disordered SiO4 tetrahedra. Electron paramagnetic resonance studies confirm the electron trapping by dynamically disordered (SiO4)4− under high energy photon illumination resulting in the formation of radical anion (SiO4)5−. The substitutional studies indicate that the [Eu3+ ← O2−] charge-transfer (CT) state is directly involved in the photoreduction process. The excitation of Sr2SiO4 ∶ Eu3+ produces the [Eu3+ ← O2−] CT state which relaxes and transfers electrons to SiO4 groups due to optically assisted rearrangement of local environment and mediates the electron transfer process to cause photoreduction of Eu3+ to Eu2+. The yellow emission is stable at room temperature and reverts to red on annealing at elevated temperature in Ar atmosphere due to thermally activated detrapping of charge carriers present at the defect centers which, in turn, convert Eu2+ to Eu3+. The thermally activated conversion of Eu2+ → Eu3+ in Sr2SiO4 is optically reversible, thereby resulting in a highly efficient material for application as an optical storage medium.

EPR investigation of Cu2+-substituted photosynthetic bacterial reaction centers: evidence for histidine ligation at the surface metal site.

The coordination environments of two distinct metal sites on the bacterial photosynthetic reaction center (RC) protein were probed with pulsed electron paramagnetic resonance (EPR) spectroscopy. For these studies, Cu2+ was bound specifically to a surface site on native Fe2+-containing RCs from Rhodobacter sphaeroides R-26 and to the native non-heme Fe site in biochemically Fe-removed RCs. The cw and pulsed EPR results clearly indicate two spectroscopically different Cu2+ environments. In the dark, the RCs with Cu2+ bound to the surface site exhibit an axially symmetric EPR spectrum with g(parallel) = 2.24, A(parallel) = 160 G, g(perpendicular) = 2.06, whereas the values g(parallel) = 2.31, A(parallel) = 143 G, and g(perpendicular) = 2.07 were observed when Cu(2+) was substituted in the Fe site. Examination of the light-induced spectral changes indicate that the surface Cu2+ is at least 23 A removed from the primary donor (P+) and reduced quinone acceptor (QA-). Electron spin-echo envelope modulation (ESEEM) spectra of these Cu-RC proteins have been obtained and provide the first direct solution structural information about the ligands in the surface metal site. From these pulsed EPR experiments, modulations were observed that are consistent with multiple weakly hyperfine coupled 14N nuclei in close proximity to Cu2+, indicating that two or more histidines ligate the Cu2+ at the surface site. Thus, metal and EPR analyses confirm that we have developed reliable methods for stoichiometrically and specifically binding Cu2+ to a surface site that is distinct from the well characterized Fe site and support the view that Cu2+ is bound at or near the Zn site that modulates electron transfer between the quinones QA and QB (QA-QB --> QAQB-) (Utschig, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemistry 37, 8278-8281) and proton uptake by QB- (Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188). Detailed EPR spectroscopic characterization of these Cu2+-RCs will provide a means to investigate the role of local protein environments in modulating electron and proton transfer.

A new high-sensitivity 10-ns time-resolution spectrophotometric technique adapted to in vivo analysis of the photosynthetic apparatus

A new spectrophotometric technique has been developed in which the absorption is sampled by short flashes produced by an optical parametric oscillator (OPO) pumped by the third harmonic of a Nd:YAG laser. Continuously tunable monochromatic flashes (6 ns duration, 1 nm bandwidth) are obtained in a domain ranging from 415 to 680 nm and 730 to 2000 nm. Light-induced absorption changes can be analyzed in a time window ranging from 10 ns to 100 s, with a 10−5 noise level. This new technique opens areas of research which are not accessible to previously existing spectrophotometers. Indeed, the high energy of the detecting flashes allows the analysis of samples of large optical density such as leaves, in the blue region of the spectrum. A few examples of application of this method to the analysis of electron transfer reactions in different types of photosynthetic apparatus (leaves, unicellular algae, and bacteria) are presented. This technique is also well suited to analyze the photodissociation of ligands associated with hemoproteins, in solution or in living cells.

Light-induced spectral changes in fully oxidized cytochrome c oxidase in the presence of oxygen.

Illumination of oxidized cytochrome oxidase with low intensity (<2 mW) light below 300 nm in the presence of oxygen causes pH-dependent spectral changes in the Soret and visible regions. The light-induced difference spectra show a peak at 438 nm and a trough at 414 nm in the Soret region and a peak at 606 nm and a shoulder at approximately 577 nm in the visible region. The effect was inhibited by cyanide, suggesting the involvement of cytochrome a3. The pH dependence indicates two titratable groups with pKa values of 6.52 +/- 0.26 and 6.85 +/- 0.15. The spectral changes are analogous to those occurring upon addition of hydrogen peroxide to the fully oxidized enzyme, which results in a mixture of species with absorbance maxima at 607 and 580 nm when referenced against the oxidized enzyme. Catalase addition affected the initial onset of the spectral change and increased the rate at which the reverse reaction occurred upon termination of illumination. The data are consistent with a mechanism involving light-induced autoreduction of the binuclear center and subsequent O2 binding, followed by the release of hydrogen peroxide and the formation of a mixture of the 607 nm and 580 nm forms.

4-Keto-bacteriorhodopsin films as a promising photochromic and electrochromic biological material

Photochromic and electrochromic spectral properties of 4-keto-bacteriorhodopsin (4-keto-BR) embedded in a polymer matrix were studied. The light-induced spectral changes were found to be similar to those for 4-keto-BR in suspension, but the duration of the photocycle is substantially longer (up to ten of h). Application of a constant electric field induces a bathochromic shift of the main absorption band, the amplitude of the field-induced spectral changes, showing a quadratic dependence on the field strength. Polymer films containing bacteriorhodopsin analogs show promise as new spectrally-selective photochromic and electrochromic materials.

Light-Induced Spectral Absorbance Changes in Relation to Photosynthesis and the Epoxidation State of Xanthophyll Cycle Components in Cotton Leaves

When cotton (Gossypium hirsutum L., cv Acaia SJC-1) leaves kept in weak light were suddenly exposed to strong red actinic light a spectral absorbance change took place having the following prominent characteristics. (a) It was irreversible within the first four minute period after darkening. (b) The difference in leaf absorbance between illuminated and predarkened leaves had a major peak at 505 nanometers, a minor peak at 465 nanometers, a shoulder around 515 nanometers, and minor troughs at 455 and 480 nanometers. (c) On the basis of its spectral and kinetic characteristics this absorbance change can be readily distinguished from the much faster electrochromic shift which has a peak at 515 nanometers, from the slow, so-called light-scattering change which has a broad peak centered around 535 nanometers and is reversed upon darkening, and from absorbance changes associated with light-induced chloroplast rearrangements. (d) The extent and time course of this absorbance change closely matched that of the deepoxidation of violaxanthin to zeaxanthin in the same leaves. (e) Both the absorbance change and the ability to form zeaxanthin were completely blocked in leaves to which dithiothreitol (DTT) had been provided through the cut petlole. DTT treatment also caused strong inhibition of that component of the 535-nanometer absorbance change which is reversed in less than 4 minutes upon darkening and considered to be caused by increased light scattering. Moreover, DTT inhibited a large part of nonphotochemical quenching of chlorophyll fluorescence in the presence of excessive light. However, DTT had no detectable effect on the photon yield of photosynthesis measured under strictly rate-limiting photon flux densities or on the light-saturated photosynthetic capacity, at least in the short term. We conclude that it is possible to monitor light-induced violaxanthin de-epoxidation in green intact leaves by measurement of the absorbance change at 505 nanometers. Determination of absorbance changes in conjunction with measurements of photosynthesis in the presence and absence of DTT provide a system well suited for future studies of meachanisms of dissipation of excessive excitation energy in intact leaves.

Light-induced spectral changes in Rose Bengal endcapped polystyrene

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTLight-induced spectral changes in Rose Bengal endcapped polystyreneS. N. Gupta, S. M. Linden, A. Wrzyszczynski, and D. C. NeckersCite this: Macromolecules 1988, 21, 1, 51–55Publication Date (Print):January 1, 1988Publication History Published online1 May 2002Published inissue 1 January 1988https://doi.org/10.1021/ma00179a012RIGHTS & PERMISSIONSArticle Views147Altmetric-Citations9LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (521 KB) Get e-Alerts Get e-Alerts

Studies on photosystem I: II. Involvement of ferredoxin in cyclic electron flow

The effects of ferredoxin (Fd) and ferredoxin-NADP reductase on the light-induced spectral changes of cytochrome f (cyt f) were investigated with specific reference to their possible involvement in the cyclic electron transfort pathway of photosystem I (PS I). The steady-state level of photooxidation of reduced cytochrome f is decreased by ferredoxin but unaffected by either ferredoxin-NADP reductase alone or ferredoxin plus ferredoxin-NADP reductase when present in equimolar concentrations. These data are taken as evidence for a cyclic electron transport pathway of: PS I → “X” → Fd → (cyt f) → PC → PS I. The reduced ferredoxin could either reduce directly plastocyanin (PC) or via cytochrome f; the data do not allow differentiation between these two possibilities. However, neither ferredoxin-NADP reductase nor cytochrome b564 appear to serve as electron carriers in this pathway.

Studies on photosystem I: I. Relationship of plastocyanin, cytochrome f and P700

Sonication of osmotically shocked chloroplasts in dilute pyridine yields a subchloroplast particle which is completely depleted of plastocyanin. Using dual-wave-length spectroscopy to study the light-induced spectral changes associated with these particles, photooxidation of reduced cytochrome f is not observed in the absence of added plastocyanin. The addition of plastocyanin, however, gives rise to a light-induced spectral change which is due solely to the oxidation of reduced cytochrome f and maximal response occurs in the presence of an equimolar amount of plastocyanin. These results are explained most simply by a strict linear sequence of electron transfer on the oxidizing side of Photosystem I from cytochrome f → plastocyanin → P700. It is found further that the presence of a detergent is required to elicit the light-induced cytochrome f oxidation signal, and that plastocyanin and the subchloroplast particles can be reconstituted by sonication. The reconstituted complex appears to be quite specific; showing a dissociation constant, during sonication, of 4.5 × 10 −7 m. This finding supports the theory that plastocyanin, in situ, is bound to the inner surface of the chloroplast membrane.

Light-induced spectral changes of P700 in the 800-nm region in Anacystis and spinach lamellae.

Abstract The light-minus-dark difference spectra of Photosystem I fragments of Anacystis and spinach lamellae in the far-red region were measured in an attempt to find the spectrum of the oxidation product of reaction center chlorophyll, P700. The results indicated a low but distinct positive band at 815 nm with a round shoulder around 750 nm. This was interpreted to be due to a P700 cation, probably P700+, from its similarity in band position and height to the chlorophyll a cation, recently observed in vitro. The molar extinction coefficient of this 815-nm band was estimated to be approximately 1.2·104 M−1·cm−1.

Light-induced absorbance changes of cytochromes and other pigments in pea chloroplasts and chloroplast fragments: I. The effect of red and near far-red actinic light and of ruby laser flashes

Light-induced spectral changes in the region 540 to 580 nm were studied by means of dual-wavelength differential spectrophotometry and by a Q-switched ruby laser flash technique in intact chloroplasts, swollen chloroplasts, and chloroplast fragments. In intact chloroplasts the major spectral components are attributed to cytochrome f (photo-oxidized), an unknown pigment with a broad band (decrease in absorbance) centered around 572 nm, and possibly a b-type cytochrome (photo-oxidized). In swollen chloroplasts and chloroplast fragments the photo-oxidation of cytochrome f is modified, cytochrome b 6 is photoreduced, and the P-572 response is diminished. The changeover from the intact to the swollen response is brought about by the addition to the suspension medium of certain cations of which sodium, with an effect beginning at about 10 m m, is the most potent. Laser flashes onto dense suspensions of high-salt chloroplasts produced fast ( t 1 2 , 300 μsec) oxidation of cytochrome f, even in the absence of ascorbate and slower ( t 1 2 , 5 msec) reduction of a b-type cytochrome. However, it is suspected that cations leaking from the chloroplasts in these dense suspensions modified the cytochrome responses.