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Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.

In this series on the application of configuration analysis (CA) to the electronic excited states of photobiological receptors, we have extended CA to two important photobiological receptors, phytochrome of photomorphogenic and biological clock systems and stentorin of the photophobic response system of stentors. In addition, the effect of substituents in chlorophylls on the electronic excitation of the chlorin and bacteriochlorin ring systems has been examined. The open-shell calculations of chlorophylls have also been performed. In the singlet excited state of phytochrome, there is a significant charge transfer from ring A to the violin moiety of the phytochrome molecule, with a tendency for the localized excitation in the A ring. The charge transfer contribution to the singlet-singlet excitation is also significant. These results are correlated with the photomorphogenic transformation of phytochrome which involves deprotonation and/or tautomerism of the ring A in the excited state. In the case of stentorin, the excitation resides largely in the aromatic system, with some charge transfer to the carbonyl groups. The photobiological significance of these results are discussed.

According to configuration analysis of the electronic state wave functions of chlorophyll a, peripheral CC(3b,4b) and isocyclic CO groups are not locally excited to any significant extent. This contrasts with the pyrone CC bond of coumarins where a substantial localization of the triplet excitation as well as photoreactions occur. The lowest excited states (1QQ and 3QQ) of chlorophyll a and bacteriochlorophyll a are also similar in correlating their state wave functions, although the ground states of these molecules are substantially different. The 11-CC bond of 11-cis-retinal protonated Schiff's bases shows no tendency for a localized excitation. Thus the primary photoexcitation of rhodopsin does not involve a preferential excitation at the 11-CC bond. The fluorescence characteristics of firefly luciferins have also been analyzed by the configuration analysis method. The most significant result is that the fluorescent states of luciferin and oxyluciferin monoanion possess a strong charge transfer character from hydroxybenzothiazole to thiazoline, in agreement with experimental observations.

It now appears that photosynthetic prokaryotes and lower eukaryotes possess higher plant phytochrome-like proteins. In this work, a second phytochrome-like gene was isolated, in addition to the recently identified Cph1 phytochrome, from the Synechocystis sp. PCC 6803, and its gene product was characterized photochemically. The open reading frame sll0821 (designated cph2 in this work) has structural characteristics similar to those of the plant phytochromes and the Synechocystis Cph1 with high amino acid sequence homology in the N-terminal chromophore binding domain. The predicted Cph2 protein consists of 1276 amino acids with a calculated molecular mass of 145 kDa. Interestingly, the Cph2 protein has two putative chromophore binding domains, one around Cys-129 and the other around Cys-1022. The Cph2 was overexpressed in E. coli as an Intein/CBD (chitin binding domain) fusion and in vitro reconstituted with phycocyanobilin (PCB) or phytochromobilin (PPhiB). Both the Cph2-PCB and Cph2-PPhiB adducts showed the typical photochromic reversibility with the difference spectral maxima at 643/690 and 655/701 nm, respectively. The Cys-129 was confirmed to be the chromophore binding residue by in vitro mutagenesis and Zn(2+) fluorescence. The microenvironment of the chromophore in Cph2 seems to be similar to that in plant phytochromes. The cph2 gene expression was dark-induced and down-regulated to a basal level by light, like the cph1 gene. These observations suggest that Synechocystis species have multiple photosensory proteins, probably with distinct roles, as in higher plants.

— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl-sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (kq) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E0. The kq (M-1 s-1) and E0 (V) values are, respectively, 7.8 times 109 and -0.134 for 1,4-benzoquinone, 8.9 times 109 and -0.309 for 1,4-naphthoquinone, 2.4 times 109 and -0.8 for nitrobenzene, 0.009 times 109 and -1.022 for azobenzene and 0 and -1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light-induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.

Time-correlated single photon counting was used to observe dynamic quenching of the hypericin and stentorin excited singlet states. The fluorescence quenching data for hypericin and stentorin were interpreted in terms of electron transfer. The observed correlation between free energy change of electron transfer and quenching rate constant suggests that quenching proceeds via electron transfer from hypericin and stentorin to the quenchers. EPR spectra for hypericin, stentorin, and stentorin chromoprotein demonstrated that free radical formation was initiated or enhanced by visible light and that similar radical species were produced in each sample. Furthermore, the EPR signal for stentorin was significantly enhanced by 1,4-benzoquinone, but the overall shape and g-value was unchanged. We suggest that electron transfer in the excited state of these chromophores results in the formation of a cation radical. This electron transfer is a rapid and efficient pathway for deactivation of hypericin and stentorin excited singlet states and should be considered when discussing the photoreactivity of hypericin as a photodynamic agent and of stentorin as the Stentor coeruleus photoreceptor.

The regulation of plant photomorphogenesis is mediated by the thermal reactions that follow light absorption by the phytochrome photoreceptor. Phytochromes are tetrapyrrolic chromoproteins that exist in two photochromically interconvertible forms, a red light absorbing species, Pr, and a far-red light absorbing form, Pfr. Upon irradiation with 670 nm light, the inactive, red light sensing Pr form is converted to the active Pfr form. Although the forward phototransformation has been studied extensively by several groups using various techniques, the Pfr--Pr photoreversion reaction that occurs upon irradiation with 730 nm light is not as thoroughly characterized. In this study, time-resolved absorption (TROD) spectroscopy is used to examine the intermediate species involved in the phytochrome photoreversion mechanism at 10 degrees C. Analysis of the TROD data identifies three species with lifetimes of 320 ns, 265 microseconds, and 5.5 ms. TROD results are described in terms of the simplest parallel and sequential kinetic models. Comparison of intermediate spectra from these mechanisms with those of previously reported species from flash photoreversion and low-temperature studies indicates that Pfr photoreversion follows a sequential pathway that does not share any intermediates with the Pr phototransformation pathway.

The photoreversible conformational change associated with the Pr-->Pfr transformation of a dicot phytochrome A (Pisum sativum, pea) has been probed by circular dichroism (CD) studies. Three different CD analysis methods have been used to determine the secondary structure of pea phytochrome A in both Pr and Pfr forms. We have shown that the secondary structure of dicot pea phytochrome A is very similar to the structure of monocot oat phytochrome A which was determined earlier [Sommer & Song (1990) Biochemistry 29, 1943-1948]. As with oat phytochrome A, an increase in the alpha-helical folding of the apoprotein takes place when photochrome in the Pr form is phototransformed to the Pfr form. This conformational change might well be a general characteristic of all phytochrome A's.

Dynamic quenching of the two lifetime component tryptophan fluorescence of Pisum phytochrome has revealed differential accessibility of certain residues. Both acrylamide and Tl+ ions showed preferential exposure of some tryptophans in Pfr-phytochrome. Greater kq's for Pfr are, however, in contrast with values for Avena phytochrome in which Pr-->Pfr conversion impedes Tl+ access. The Pr short lifetime component was more accessible to Cs+; however, the long component accessibility was approximately 2-fold higher in Pfr. 2-Hydroxy-5-nitrobenzyl bromide (HNB-Br) modification of native Pisum phytochrome was used to reduce the total number of fluorescent tryptophans. The absence of the fluorescence contributions of the three residues which reacted with HNB-Br in both photoisomers increased the Tl+ Ksv's for Pr and Pfr. The two additional HNB-Br modifications specific for Pfr resulted in a reversal of the Stern-Volmer plots relative to the unmodified protein. The regions around four of the 10 tryptophans may represent conformationally photoresponsive areas in Pisum phytochrome A. Furthermore, topographic changes associated with the phytochrome phototransformation are not confined to the 58-kDa chromphore domain, and they involve most if not all of the region from Trp-365 to Trp-787. We also provide evidence that the protein conformation in this region is not completely conserved between Pisum and Avena phytochromes.

Polarized absorption difference profiles have been obtained with 3-6-ps resolution between 565 and 630 nm for the chromoproteins stentorin I and stentorin II, isolated from the protozoan ciliate Stentor coeruleus. Control experiments were performed on hypericin and on the stentorin chromophore in methanol. The isotropic decays of stentorin I and the stentorin chromophore are dominated by components with lifetimes conservatively longer than 1 ns. Stentorin I, the stentorin chromophore, and hypericin all develop, within 0.4 and

Absorption difference profiles were obtained at wavelengths from 640 to 700 nm with 1-2-ps resolution in a study of primary photoprocesses in the Pr-->Pfr transformation in native oat phytochrome. These experiments were performed using low-intensity laser pulses at high repetition rate; fast sample recycling ensured that essentially all phytochrome species were excited from the Pr ground state. The Pr*-stimulated emission decay at wavelengths > 670 nm exhibits major components with lifetimes of approximately 16 and 50-60 ps. Formation of the asymptotic 695-nm lumi-R absorption spectrum rapidly follows stimulated emission decay. Photoexcitation of one or both of the lumi-R intermediates instantaneously recreates fluorescing Pr* phytochrome, which is spectroscopically and kinetically indistinguishable from that generated by direct illumination of ground-state Pr. This is consistent with assignment of lumi-R as a species in which the chromophore has isomerized from the Z,Z,Z to the Z,Z,E conformation. Anisotropy studies indicate that the orientations of the Pr and lumi-R absorption transition moments are nearly parallel, since little anisotropy decay occurs during the 500-ps time window of these experiments.

Hypericin (Hyp), the parent chromophore of the photoreceptor protein (stentorin) participates in photophobic movements in Stentor coeruleus. The photoreactivity in the lowest singlet excited state (S 1 ) of hypericin has been studied with respect to the solvent effects on absorption and fluorescence spectra and fluorescence decay kinetics. The primary photoreaction of hypericin was probed through (1) hydrogen-bonding effect, (2) deuterium solvent isotope effect, (3) pH dependence and (4) fluorescence quenching by p-benzoquinone

A novel antibody-fluorescence method has been developed to elucidate the chromophore topography in phytochrome as it undergoes a photochromic transformation. Forster energy transfer from N-terminal bound, fluorescently labeled Oat-25 Fab antibody fragments to the phytochrome chromophore was measured. The results suggest that the chromophore moves relative to the N-terminus upon the Pr-->Pfr phototransformation. This conclusion is consistent with previous models which have proposed a reorientation and an interaction of the Pfr chromophore with the N-terminus. The method described appears to be the first study of a Forster energy transfer measurement using a donor-label attached to a Fab fragment of a photosensor protein.

The effect of the singlet oxygen quencher crocetin on the photomotile responses and photodynamic killing of the ciliated protozoan Blepharisma japonicum was investigated. The percentage of cells surviving photodynamic killing is higher in the presence of the water-soluble carotenoid derivative than in control samples. However, no significant difference is observed in the photobehavior of the control and crocetin-containing samples. These results indicate that blepharismin-sensitized photodynamic reactions occur in Blepharisma, but do not play any meaningful role in initiating photosensory processes.

Stentorin serves as the photoreceptor for the photophobic and negative phototactic responses in Stentor coeruleus. Two forms of the stentorin have been isolated and purified. The strongly fluorescent form, stentorin I at pH 7.8, exhibited nearly exponential fluorescence decay monitored at 620 nm, having two comparable lifetime decay components of 2.53 ns (47%) and 5.95 ns (53%). Stentorin I showed no significant time-resolved fluorescence emission spectra in the picosecond-nanosecond time scales. The weakly fluorescent form, stentorin II, exhibited an ultrafast fluorescence decay component (10 ps) at an emission wavelength of 630 nm and pH 7.8. The amplitudes of the multi-component fluorescence in stentorin II were found to be emission wavelength-dependent. Furthermore, the fluorescence emission spectrum was time-resolvable in the picosecond time scales. Effects of pH and pD on the fluorescence decay kinetics and time-resolved spectra of stentorins I and II have also been investigated. Results are suggestive of proton dissociation as a primary photoprocess from the excited state of stentorin II.

We have studied the effects of pH, ionic strength, and hydrophobic fluorescence probes, 8-anilinonaphthalene-1-sulfonate (ANS) and bis-ANS, on the structure of intact (124-kDa) Avena phytochrome. The Pfr form of phytochrome forms oligomers in solution to a greater extent than the Pr form. Hydrophobic forces play a major role in the oligomerization of phytochrome, as suggested by fluorescence and monomerization by bis-ANS. However, electrostatic charges also take part in the phytochrome oligomerization. The partial proteolytic digestion patterns for the Pr and Pfr species are different, but binding of bis-ANS to the phytochrome abolishes this difference and yields an identical proteolytic peptide mapping for both spectral forms of phytochrome. This appears to result from bis-ANS binding at the carboxy-terminal domain, which induces monomerization of phytochrome oligomers. A second bis-ANS binding at an amino-terminal site blocks cleavage sites of trypsin and alpha-chymotrypsin. Bis-ANS especially blocks access of the proteases to the amino-terminal cleavage site that produces an early proteolytic product (114/118 kDa) on SDS gels. The bis-ANS binding does not, however, affect the proteolytic cleavage site that occurs in the hinge region between the two structural domains of phytochrome, the chromophore domain and the C-terminal non-chromophore domain. A chromophore binding site in the Pfr form is apparently exposed for preferential binding of bis-ANS, causing cyclization of the chromophore and bleaching of its absorbance at 730 nm. These observations have been discussed in terms of a photoreversible topographic change of the chromophore/apoprotein during the phototransformation of phytochrome.

The relative extent of chromophore exposure of the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of 124-kDa oat phytochrome and the secondary structure of the phytochrome apoprotein have been investigated by using zinc-induced modification of the phytochrome chromophore. The absence of bleaching of Pr in the presence of a 1:1 stoichiometric ratio of zinc ions, in contrast to extensive spectral bleaching of the Pfr form, confirms previous reports of differential exposure of the Pfr chromophore relative to the Pr chromophore. The emission of orange fluorescence by zinc-chelated Pfr indicates that the Pfr chromophore has been modified from its native extended/semi-extended conformation to a cyclohelical conformation. Circular dichroism (CD) analyses of native phytochrome in 20 mM Tris buffer suggests that the Pr-to-Pfr phototransformation is accompanied by a photoreversible change in the far-UV region consistent with an increase in the alpha-helical folding of the apoprotein. The secondary structure of phytochrome in Tris buffer, as determined by CD, differs slightly from that of phytochrome in phosphate buffer, suggesting that phytochrome is a conformationally flexible molecule. Upon the addition of a 1:1 molar ratio of zinc ions to phytochrome, a dramatic change in the CD of the Pfr form is observed, while the CD spectrummore » of the Pf form is unaffected. Analysis of the bleached Pfr CD spectrum by the method of Chang et al. (1978) reveals that chelation with zinc ions significantly alters the secondary structure of the phytochrome molecule, specifically by increasing the beta-sheet content primarily at the expense of alpha-helical folding.« less

Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr left arrow over right arrow Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.

Time-resolved circular dichroism spectroscopy in the far-UV spectral region was used to examine the intermediates of the phytochrome photoreversion reaction (Pfr --> Pr). Three intermediates, lumi-F (tau = 320 ns), meta-Fa (tau = 265 micros) and meta-Fb (tau = 5.5 ms), have been identified in a simple sequential kinetic photoreversion mechanism by absorption spectroscopy [Linschitz, H., Kasche, V., Butler, W. L., & Siegelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403; Pratt, L. H., & Butler, W. L. (1968) Photochem. Photobiol. 8, 477-485; Burke, M., Pratt, D. C., & Moscowitz, A. (1972) Biochemistry 11, 4025-4031; Spruit, C. J. P., Kendrick, R. E., & Cooke, R. J. (1975) Planta (Berlin) 127, 121-132; Eilfeld, P., & Rudiger, W. (1985) Z. Naturforsch. 40c, 109-114; Chen, E., Lapko, V. N., Lewis, J. W., Song, P.-S., & Kliger, D. S. (1996) Biochemistry 35, 843-850]. In order to correlate the unfolding of the N-terminal alpha-helical segment with one or more of the intermediate species, time-resolved methods were coupled with the structurally sensitive probe of CD in the far-UV spectral region. Analysis of the TRCD data associates the decrease in alpha-helical content that occurs upon formation of Pr with decay of the meta-Fa intermediate. This unfolding process occurs with a time constant of 310 +/- 125 micros, which is consistent with the 265-micros lifetime for meta-Fa.

Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomyces cerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R. (1990) Z. Naturforsch, 45c, 987-998; Parker, W., et al. (1993) Bioconjugate Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to the lyase activity. It appears that the role of the five amino acid residues in the N- and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.

Phytochrome is a tetrapyrrole chromoprotein. It serves as a sensitive photosensor for red lightmediated gene expression and other developmental/morphological responses in plants. In this paper photochemical dynamics of the phytochrome molecule have been described in terms of photoisomerization of the tetrapyrrole chromophore in its singlet excited state and subsequent thermal processes in the Pr Pfr phototransformation of phytochrome. Stentorin acts as the photosensor molecule in the ciliate Stentor coeruleus. This unicellular protozoan is most sensitive to red light (610-620 urn). Stentor also senses the direction of light propagation as evidenced by their light-avoiding and negative phototactic swimming behaviors. This aneural photosensory phenomenon is triggered by the photoreceptor stentorin. The possible involvement of a light-induced transient proton release from the photoreceptor as the primary mechanism of light-signal processing has been discussed on the basis of picosecond fluorescence decays and time-resolved fluorescence spectra of stentorin in solution. An initial sensory signal generated by the primary photoprocess of stentorin then triggers subsequent transduction steps that include calcium ion influx from the extracellular medium. Calcium ion influx from the extracellular medium to the cytosol causes the Stentor cell to reverse its ciliary beating and subsequently steer away from the light trap. II.

The chemical reactivity of phytochrome chromophores (Pr and Pfr forms) has been calculated by the frontier electron and perturbation methods. Results suggest that the major reactivity of the Pr and Pfr chromophores of phytochrome remain essentially identical upon the phototransformation of phytochrome. The nucleophilic reactivity of the Pr phytochrome chromophore is highest at the central methine carbon (position-10), whereas the electrophilic reactivity is generally spread over the tetrapyrrole chromophore. These results are consistent with the phytochrome model that the Pfr chromophore is much more exposed than is the Pr chromophore.

— Electronic spectra of indoles are very sensitive to solvents. These solvent effects are particularly pronounced in the case of the fluorescence spectra of indoles. In order to elucidate the solvent band shifts during the relaxation of the excited states, particularly in polar solvents, the dipole moments of the excited singlet states have been estimated from the data of solvent-dependent Stokes shifts. In addition to indole, indole-5-carboxylic acid, indole-3-carboxylic acid, indole-2-carboxylic acid, 5-cyano- and 5-bromo-indoles have been investigated. All indoles showed a substantial increase in dipole moment upon excitation to the emitting state. These results are generally consistent with the Pariser-Parr-Pople SCF MO calculations.

The reactivity of the phytochrome chromophore and related tetrapyrroles towards ozone and tetranitromethane was investigated. Both oxidizing reagents cause bleaching of the main absorp­tion band of the pigment. The rate constants for this bleaching were determined under conditions of pseudo first order reaction kinetics. The rate constants for the reaction with ozone are similar for native phytochrome and for freely accessible tetrapyrroles (biliverdin, small chromopeptides from phytochrome) indicating that accessibility is not the limiting factor for the reaction with ozone. Under a variety of conditions, the Pfr chromophore reacts by about 10% faster than the Pr chromophore. This may reflect the true difference in reactivity. The rate constants for the reaction with tetranitromethane are much larger for biliverdin, bilirubin and small chromopeptides from phytochrome than for native phytochrome. The limiting factor for this reaction in native phytochrome therefore is the accessibility of the chromophore by the reagent. Previous conclusions on the difference in exposure of the tetrapyrrole chromophore in Pr and Pfr are confirmed.

Heavy atom salts (e.g., KI, LiBr, KCl) and solvents do not quench fluorescence of diphenyl polyenes and retinol, whereas the fluorescence of all-trans retinal is enhanced by alkali salts. The enhanced fluorescence is attributable to the effect of cation counterions which complex with the retinal carbonyl to enlarge the gap between the π, π* and n, π* states. Retinol and retinal show significant temperature dependence of Φf (14–120 K), suggesting an efficient torsional relaxation. On the other hand, phytochrome shows essentially temperature-independent Φf in 14–200 K. At 298 K, Φf becomes ⩽ 10-4, and the rate constant for the primary radiationless process is ≈ 1012 s-1.

The carcinogenicity of 8-methoxypsoralen (8-MOP) and aflatoxin B 1 (AFB 1 ) with longwave ultraviolet light (UVA) to hairless mouse skin was investigated. Skin tumors were induced efficiently by 8-MOP + UVA, and the time to 50% tumor incidence was about 24 weeks. Histopathologically some tumors were squamous cell carcinomas. AFB 1 did not show any phototoxic and photocarcinogenic effects on mouse skin in this study. Although the structure and the photoreactivity of AFB 1 to DNA were similar to those of 8-MOP, the photocarcinogenic response of these compounds to mouse skin was quite different.

Summary Chromophore oxidation using tetranitromethane was performed with 124 kDa, 59kDa, and 39kDa oat phytochrome in both photoreversible forms (Pr and Pfr). Kinetics and intermediates of oxidation were analyzed using a diode array spectrophotometer. A transient species (λmax = 580 and 505 nm) was observed exclusively from the Pfr (or Pbl in the case of 39 kDa phytochrome) forms of degraded phytochromes. This oxidation product was isolated by rapid size-exclusion HPLC. Comparison of its absorption spectrum with those of model compounds revealed a strong similarity to biladienes-a,b, indicating that the primary attack of tetranitromethane takes place at the methine bridge, predominantly at C-15. This is interpreted in terms of a different accessibility of carbon-15 of the chromophore in the Pr vs. Pfr form.

The application of surface-enhanced resonance Raman scattering (SERRS) spectroscopy to the analysis of the configuration of biliverdin dimethyl ester (BVDE) is reported. SERRS spectra obtained by adsorption of the compounds onto an electrochemically roughened silver electrode and recorded at 7 K were intense and free of significant photodegradation. The similarity of the SERRS and resonance Raman (RR) spectra obtained under identical conditions suggests that no perturbation of the electronic structure of the BVDE occurs upon interaction with the silver surface, and that the distribution of conformers comprising the BVDE solution is not changed. SERRS spectra of the deuterated and monoprotonated Z,Z,Z isomer are also presented. To investigate the influence of configuration upon the Raman spectrum we have synthesized and purified the E,Z,A, and Z,Z,E isomers of BVDE. Excellent SERRS spectra were obtained from the solutions of the compounds eluted directly from the TLC plates.

Enzymatically generated triplet acetone transfers its energy to the ground state phytochrome and promotes to some extent, in the dark, the conversion of Pr into Pfr and of Pfr into Pr. This is the first report of inverse dark reversion “in vitro”.

1. 1. The peridinin·chlorophyll a ·protein complex from Amphidinium carterae (Plymouth 450) shows spectroscopic characteristics (absorption, CD, fluorescence polarization, lifetime and energy transfer) essentially identical with peridinin·chlorophyll a ·protein complexes from Glenodinium sp., Gonyaulax polyedra and Amphidinium rhyncocephaleum . 2. 2. The apoprotein of peridinin·chlorophyll a ·protein complexes is globular, with an isotropic rotational relaxation time (e.g. 33 ns for the A. caterae peridinin·chlorophyll a ·protein), as deduced from the dynamic depolarization data. 3. 3. The chromophores (4 peridinins and 1 chlorophyll a for peridinin·chlorophyll a ·protein complexes from Glenodinium sp., G. polyedra and A. rhyncocephaleum and 9 and 2, respectively, for peridinin·chlorophyll a ·protein of A. carterae ) are accommodated in a hydrophobic crevice and not exposed to the solvent. The surface of the protein is highly hydrophilic. 4. 4. No evidence for chlorophyll-chlorophyll interactions in the A. carterae peridinin·chlorophyll a ·protein was obtained. This implies that binding crevices for two chlorophylls and half of peridinins (four to five) are located at some distance from each other. 5. 5. The peridinin·chlorophyll a ·protein complexes function as the photosynthetic antenna pigment. In addition, peridinins effectively protect chlorophyll a from photodecomposition.

1. 1. Several vitamin B12 derivatives including descobalt B12 show unusual inversion of their CD signs upon rapid cooling to liquid N2 temperature, although the room temperature CD signs are conserved by a slower cooling to the same temperature. As a possible explanation for this puzzling observation, a micro-environmental birefringence around the chromophore imbedded in an organic glass is proposed. 2. 2. Absorption, fluorescence, phosphorescence, and polarization spectra of descobalt B12 can be correlated with those of porphyrin free bases, as these two molecular systems share many similarities in their electronic structire. 3. 3. Molecular orbital calculations of polarization directions further support the analogy between the spectroscopic characteristics of corrins and porphyrins, and are generally in good agreement with the fluorescence polarization data.

— The order of inhibition of the photooxidation of chlorophyll a in ethanol and ethanol-benzene is as follows: β-carotene, α-tocopherol, benzoquinone, DABCO, menadione, cholesterol and KI. The quenching of singlet oxygen by β-carotene occurs by a collisional quenching mechanism with a diffusion-controlled rate of 1.7 × 1010M-1 s-1. Photodecomposition of Chi a is faster in ethanol-D2O than in ethanol-H2O. Photoirradiation (660 nm) of the peridinin-Chl a-protein complex, a photosynthetic light-harvesting pigment isolated from marine dinoflagellates, did not show any photo-decomposition of its Chi a in H2O or D2O, even after an extended period (12 h) of irradiation. However, the carotenoid, peridinin, in the photosynthetic antenna pigment was photobleached (ca. 10%) during the irradiation. We conclude that the singlet oxygen formed as a result of the Chi photosensitization is immediately quenched by the low-lying triplet state of four peridinin molecules (per Chl a) bound within the same protein crevice. The carotenoid thus effectively protects Chl a from photodynamic damage, providing a direct proof for the protective role of carotenoids in the photosynthetic pigment complex.

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.

The 360 MHz proton NMR spectra of the Pr and Pfr forms of phytochrome have been recorded to probe the nature of the phytochrome phototransformation (Pr → Pfr). The NMR spectra of aliphatic protons in Pr and Pfr proteins are similar, suggesting that the conformation of both proteins are not drastically different. However, the NMR spectrum of aromatic and the -NH- proton resonance region of Pfr differs significantly from that of Pr, including the absence of a resonance at 6.15 ppm in the former. Differences in the NMR spectra of small and large mol wt phytochromes have also been noted.

— To probe the nature of primary photoprocess and the mechanism of the phototransformation of undegraded 124 kDa oat phytochrome, solvent deuterium isotope effects on the fluorescence and phototransformation of phytochrome have been investigated. The fluorescence intensity and lifetime of phytochrome (Pr form) are greater in D20-buffer than in H2O-buffer, suggesting a possible involvement of proton transfer in the primary photoprocess of phytochrome. Although the photostationary equilibrium (Pr to Pfr ratio) was not altered by deuterium oxide, in contrast to degraded phytochrome, the rate constants of both transformations, Pr Pfr and Pfr Pr were enhanced by up to 24%. The Pr to Pfr phototransformation of degraded phytochrome, however, was retarded by about the same percentage in D2O. These opposite effects of D2O with degraded and undegraded phytochromes underscore the fact that the Pr form from the former reverts to the Pr form in the dark, apparently catalyzed by deuterated general and/or conjugate acidic group(s). With the degraded phytochrome the deuterium oxide enhancement of the rate of dark reversion was approximately 2-fold (Sarkar and Song, 1981). Both the fluorescence intensity and the rates of phototransformation of phytochrome were enhanced in D2O with successive photocyclings (Pr Pfr Pr Pfr Pr etc.) with alternating red and far-red irradiation. It has been proposed that successive photocycling of phytochrome in D2O results in proton-deuteron exchange in the partially exposed Ptr chromophore and/or its surrounding amino acid residues.

— The blue light absorption band of roseoflavin is polarized along the axis roughly connecting N3-C8 positions. A weak, second ππ* transition with a polarization angle of ca. 25° is hidden under the short wavelength side of the blue absorption band. The excited state of roseoflavin is somewhat more basic than the ground state, by a 1.5 pKa unit. The fluorescence quantum yield and lifetime of roseoflavin are substantially lower than those of other flavins, thus making it kinetically less efficient as a blue light photoreceptor.

— 8–Methoxypsoralen (8–MOP) is shown to form a covalent conjugate with bovine serum albumin (BSA) by UVA irradiation in the presence of 02. The photoreaction is shown to involve oxidation of 8–MOP itself as a first step, producing an oxidation product which reacts readily with protein. Thus, this photoreaction appears to be completely different from the known 8–MOP photo-cycloaddition reaction with DNA. Among several proteins studied, human serum albumin, histone type 11, RNAse A and lysozyme also undergo photoinduced addition by 8–MOP. Chemical modifications of various amino acid residues in BSA revealed tyrosine-OH as one of the reaction sites. Irradiation (12 h) with UVA at 1.78 J/ds intensity resulted in approximately 1.5 mot of 8–MOP bound to 1 mol of BSA.

The photosynthetic light-harvesting complex, peridinin-chlorophyll a-protein, was isolated from several marine dinoflagellates including Glenodinium sp. by Sephadex and ion-exchange chromatography. The carotenoid (peridinin)-chlorophyll a ratio in the complex is estimated to be 4:1. The fluorescence excitation spectrum of the complex indicates that energy absorbed by the carotenoid is transferred to the chlorophyll a molecule with 100% efficiency. Fluorescence lifetime measurements indicate that the energy transfer is much faster than fluorescence emission from chlorophyll a. The four peridinin molecules within the complex appear to form two allowed exciton bands which split the main absorption band of the carotenoid into two circular dichronic bands (with negative ellipticity band at 538 nm and positive band at 463 nm in the case of peridinin-chlorophyl a-protein complex from Glenodinium sp.). The fluorescence polarization of chlorophyll a in the complex at 200 K is about 0.1 in both circular dichroic excitation bands of the carotenoid chromophore. From these circular dichroic and fluorescence polarization data, a possible molecular arrangement of the four peridinin and chlorophyll molecules has been deduced for the complex. The structure of the complex deduced is also consistent with the magnitude of the exciton spliting (ca. greater than 3000 cm-1) at the intermolecular distance in the dimer pair of peridinin (ca. 12 A). This structural feature accounts for the efficient light-harvesting process of dinoflagellates as the exciton interaction lengthens the lifetime of peridinin (radiative) and the complex topology increases the energy transfer probability. The complex is, therefore, a useful molecular model for elucidating the mechanism and efficiency of solar energy conversion in vivo as well as in vitro.

— 5,7-DimethoxJtcoumarin (DMC) dimerizes through the C4-photo-cycloaddition of 3,4-double bonds to form a syn head-to-tail dimer on direct irradiation (Λ≥ 300 nm) in acetonitrile or benzene solution. The quantum yield of the photocyclodimerization in acetonitrile is 0.068 which is greater than that of coumarin. In the presence of triplet sensitizers such as benzophenone, 5,7-dimethoxycoumarin forms an anti dimer with the quantum yield greater than 0.08. The structure of the photodimers has been elucidated by IR, UV, NMR, and mass spectrometry. The results of luminescence studies, triplet quenching and sensitization revealed that the syn head-to-tail dimer was formed via an excited singlet precursor, while the anti dimer was formed via the excited triplet state.

Surface-enhanced resonance Raman scattering (SERRS) spectra of phytochrome at 77 K are reported. The spectra reveal significant differences between Pr and Pfr forms of phytochrome. SERRS spectra of C-phycocyanin Z,Z,Z- and Z,Z,E-chromopeptide isomers at 77 K are also reported. The phycocyanin chromopeptide studies are used to provide a basis for interpreting the phytochrome SERRS spectra. The spectra indicate that photoisomerization of chromophores from C-phycocyanin chromopeptides (from a Z,Z,Z to a Z,Z,E configuration) is detectable with SERRS.

— Significant spectroscopic difference was found between the 3(π, π*) state of coumarins and the 3La state of benzpyrene. No analogy of the partially localized triplet state associated with the coumarin chromophore was revealed in the case of the potent carcinogen, benzo[a]pyrene. Instead, the 3La state of benzo[a]pyrene is characterized by more derealization than that of the non–carcinogenic benzo[e]pyrene. Therefore, the predominant photoreaction between benzo[a]pyrene and DNA bases does not seem to involve cycloaddition in contrast to the coumarin-pyrimidine system. Reactivity indices have been calculated, and results are consistent with recent experimental findings. In addition, spectroscopic properties of benzo[a]pyrene and benzo[e]pyrene have been comparatively described in terms of relatively high-resolution spectra, polarization measurements, and molecular orbital calculations.