FLUORESCENCE SPECTROSCOPY OF A P700-CHLOROPHYLL-PROTEIN COMPLEX*

Abstract. Chlorophyll-protein complexes enriched in the Photosystem I reaction center chlorophyll (P700) exhibit a fluorescence emission maximum at 696 nm at - 196°C The height of this 696 nm emission relative to the emission at 683 nm from antenna chlorophyll a increases proportionally with the P700 concentration while the total fluorescence yield of the complex decreases. The 696 nm emission could possibly be from an absorbing form of antenna chlorophyll a that may be somewhat enriched along with P700 in Photosystem I fractions. However, evidence resulting from glycerol treatment which appears to decrease the rate of resonance energy transfer between antenna chlorophyll and P700 favors the hypothesis that the emission comes from a photooxidized P700 dimer (Chl⁺-Chl) absorbing near 690 nm. In turn, this fluorescence evidence provides additional support for the model of a P700 dimer involving exciton interaction. Absorption in the wavelength region of 450 nm specifically excites emission at 696 nm from the P700-chlorophyll complex.     

LIQUID-CRYSTALLINE PHASE TRANSITION OF MONOMOLECULAR LAYERS OF CHLOROPHYLL a*

Monomolecular layers of chlorophyll a at the air-water interface were investigated by absorption spectroscopy and simultaneous thermodynamic measurements. Whereas at temperatures near 20°C at all pressures, only a liquid phase exists, at a temperature of 4°C, a liquid-crystalline phase transition is observed at a surface pressure of 5 dynes/cm. Pressure-induced changes in the chlorophyll arrangement become evident from a drastic change in the absorption spectra, accompanying the phase transition. The crystalline phase exhibits an extremely narrow absorption band (halfwidth below 9 nm) centered at 698 nm, indicative of a coplanar chlorophyll arrangement in a well-ordered environment. It is highly probable that in these model membranes a chlorophyll arrrangement could be established that is equivalent to the one proposed for the reaction center P700.     

LIGHT-ABSORPTION AND ELECTRON-TRANSPORT BALANCE BETWEEN PHOTOSYSTEM II AND PHOTOSYSTEM I IN SPINACH CHLOROPLASTS

Abstract— The distribution of absorbed light and the turnover of electrons by the two photosystems in spinach chloroplasts was investigated. This was implemented upon quantitation of photochemical reaction centers, chlorophyll antenna size and composition of each photosystem (PS), and rate of light absorption in situ. In spinach chloroplasts, the photosystem stoichiometry was PSIIJPSII_α/PSII_β/PSI= 1.3/0.4/1.0. The number (N) of chlorophyll (a+b) molecules associated with each PS was N(PSII_α)/N(PSII_β)/N(PSI)=230/100/200, i.e. about 65% of all Chl is associated with PSII and about 35% with PSI. Light absorption by PSII in vivo is selectively attenuated at the molecular, membrane and leaf levels, (a) The rate of light absorption by PSII was only 0.85 that of PSI because of the lower rate of light absorption by Chl b as compared to Chl a (approximately 80% of all Chl b in the chloroplast is associated with PSII). (b) The exclusive localization of PSII_α in the membrane of the grana partition regions and of PSI in intergrana lamellae resulted in a differential “sieve effect” or “flattening of absorbance” by the photosystems in the two membrane regions. Due to this phenomenon, the rate of light absorption by PSII was lower than that of PSI by 15-20%. (c) Selective filtering of sunlight through the spinach leaf results in a substantial distortion of the effective absorbance spectra and concomitant attenuation of light absorption by the two photosystems. Such attenuation was greater for PSII than for PSI because the latter benefits from light absorption in the 700-730 nm region. It is concluded that, in spite of its stoichiometric excess in spinach chloroplasts, light absorption by PSII is not greater than that by PSI due to the different molecular composition of the two light-harvesting antenna systems, due to the localization of PSII in the grana, and also because of the light transmission properties through the leaf. The elevated PSII/PSI reaction center ratio of 1.7 and the association of 65% of all Chl with PSII help to counter the multilevel attenuation of light absorption by PSII and ensure a balanced PSII/PSI electron turnover ratio of about 1:1.     

AUTHENTIC in vivo ABSORPTION SPECTRA FOR CHLOROPHYLL IN LEAVES AS DERIVED FROM in situ AND in vitro MEASUREMENTS*

On the basis of a comprehensive treatment of light propagation in plant tissue the first authentic in vivo absorption spectrum of chlorophyll in a leaf (of Catalpa bignonioides) has been elucidated. An experimental-theoretical procedure is described, which permits derivation of the authentic chlorophyll spectrum by purifying the in vivo absorption spectrum of a leaf from the distortions of optical origin: multiple scattering and distributional error. The procedure involves measurements of internal and external light fluxes, spatial characterization of tissue layers, cells, chloroplasts and grana stacks. Furthermore, one has to determine the local chlorophyll concentrations. The theories of radiation transfer and absorption statistics make up the theoretical basis of the procedure. The derived authentic chlorophyll spectrum differs both from the in vivo spectrum of the leaf and (although to a smaller degree) from the in vitro spectrum of chlorophyll-protein complexes extracted from the same leaf.     

CHLOROPHYLL-PROTEINS AND REACTION CENTER PREPARATIONS FROM PHOTOSYNTHETIC BACTERIA,ALGAE AND HIGHER PLANTS*,‡

Abstract— The use of sodium dodecyl sulfate to dissociate photosynthetic membranes followed by standard fractionation techniques yields chlorophyll-proteins and reaction center complexes with molecular weights of 500,000 or less. Much about the structure and function of photo-synthetic units in vivo can be deduced from the properties of the isolated complexes. The Bchl-protein from green bacteria is approximated by an incompletely filled sphere ? 80 Â in diameter consisting of four identical subunits. The five Bchl molecules in each subunit are 14 to 20Â apart. The related Chl a-proteins from a blue-green alga and various eukaryotic plants may have similar structural characteristics. The Chl a-protein from a blue-green alga contains one molecule of P700 per 60–90 Chl a molecules. The quantum requirement for P700 oxidation is 2.6 or less. The midpoint potential in various preparations ranges from 0.38 V to 0.42 V. Green algae and higher plants yield a Chl a-protein similar to that from the blue-green alga; in addition they yield another Chl-protein (mol. wt. = 2–3×10⁴) which contains an equal amount of Chl a and Chl b. These two Chl-proteins account for most of the chlorophyll in these organisms. Two photosynthetic bacteria (Rhodopseudomonas viridis and Chromatium) yield protein complexes containing Bchl, carotenoid, and bound cytochromes. The reaction center complex from R. viridis contains P960 (E_m, ₈= 0.39 to 0.42 V), cytochrome 558 (E_m,8= 0.33 V) and cytochrome 553 (E_m,7=— 0.02 V). Quantum requirements for P960 and C558 oxidation are ?2.2 and 3.0, respectively. Complex A from Chromatium contains Bchl 890, P883, cytochrome 556 (E_m,8= 0.34 V) and cytochrome 552 (E_m,7=?0.04 V). The quantum requirement for C556 oxidation is about 15. Both high- and low-potential cytochromes can donate electrons to the reaction center chlorophyll present in either complex. This fact supports the idea that only one kind of photochemical reaction center functions in photosynthetic bacteria. An hypothesis about the nature of the photosynthetic unit in purple bacteria is outlined.     

Primary processes in plant photosynthesis: photosystem I reaction center

The photosystem I (PSI) pigment-protein complex of plants converts light energy into a transmembrane charge separation, which ultimately leads to the reduction of carbon dioxide. Recent studies on the dynamics of primary energy transfer, charge separation, and following electron transfer of the reaction center (RC) of the PSI prepared from spinach are reviewed. The main results of femtosecond transient absorption and fluorescence spectroscopies as applied to the P700-enchied PSI RC are summarized. This specially prepared material contains only 12–14 chlorophylls per P700, which is a special pair of chlorophyll a and has a significant role in primary charge separation. The P700-enriched particles are useful to study dynamics of cofactors, since about 100 light-harvesting chlorophylls are associated with wild PSI RC and prevent one from observing the elementary steps of the charge separation. In PSI RC energy and electron transfer were found to be strongly coupled and an ultrafast up-hill energy equilibration and charge separation were observed upon preferential excitation of P700. The secondary electron-transfer dynamics from the reduced primary electron acceptor chlorophyll a to quinone are described. With creating free energy differences (ΔG₀) for the reaction by reconstituting various artificial quinones and quinoids, the rate of electron transfer was measured. Analysis of rates versus ΔG₀ according to the quantum theory of electron transfer gave the reorganization energy, electronic coupling energy and other factors. It was shown that the natural quinones are optimized in the photosynthetic protein complexes. The above results were compared with those of photosynthetic purple bacteria, of which the structure and functions have been studied most.     

ELECTROPHORETIC AND SPECTROPHOTOMETRIC STUDIES OF CHLOROPHYLL-PROTEIN COMPLEXES FROM TOBACCO CHLOROPLASTS. ISOLATION OF A LIGHT HARVESTING PIGMENT PROTEIN COMPLEX WITH A MOLECULAR WEIGHT OF 70,000

Abstract— …After a short-term solubilization with sodium dodecyl sulphate, chloroplast membranes of tobacco were separated by polyacrylamide gel electrophoresis into three chlorophyll-protein complexes. In addition to the two major complexes termed I and II_c corresponding respectively to P700 chlorophyll a -protein and light-harvesting chlorophyll a/b -protein described by Thornber (1975), a relatively stable complex termed II_a has been observed. This new complex has an apparent molecular weight of 70,000 daltons and possesses Chl a and b.
Complexes I, II_a and II_C have been isolated and precise spectroscopic analyses have been performed. Fourth derivative analyses of low temperature absorption spectra suggest that complex II_a seems more representative than II_C of chlorophyll a forms present in intact thylakoid membranes.
Moreover, the electrophoretic study reveals that CP_I and CP_II are composed of only one polypeptidic subunit with respective molecular weights of 68,000 and 24,000 daltons.     

Quenching of chlorophyll a singlets and triplets by carotenoids in light-harvesting complex of photosystem II: comparison of aggregates with trimers

Laser-induced changes in the absorption spectra of isolated light-harvesting chlorophyll a/b complex (LHC II) associated with photosystem II of higher plants have been recorded under anaerobic conditions and at ambient temperature by using multichannel detection with sub-microsecond time resolution. Difference spectra (ΔA) of LHC II aggregates have been found to differ from the corresponding spectra of trimers on two counts: (i) in the aggregates, the carotenoid (Car) triplet–triplet absorption band (ΔA>0) is red-shifted and broader; and (ii) the features attributable to the perturbation of the Q_y band of a chlorophyll a (Chla) by a nearby Car triplet are more pronounced, than in trimers. Aggregation, which is known to be accompanied by a reduction in the fluorescence yield of Chla, is shown to cause a parallel decline in the triplet formation yield of Chla; on the other hand, the efficiency (100%) of Chla-to-Car transfer of triplet energy and the lifetime (9.3 μs) of Car triplets are not affected by aggregation. These findings are rationalized by postulating that the antenna Cars transact, besides light-harvesting and photoprotection, a third process: energy dissipation within the antenna. The suggestion is advanced that luteins, which are buried inside the LHC II monomers, as well as the other, peripheral, xanthophylls (neoxanthin and violaxanthin) quench the excited singlet state of Chla by catalyzing internal conversion, a decay channel that competes with fluorescence and intersystem crossing; support for this explanation is presented by recalling reports of similar behaviour in bichromophoric model compounds in which one moiety is a Car and the other a porphyrin or a pyropheophorbide.     

CHLOROPHYLL a FLUORESCENCE OF GONYAULAX POLYEDRA GROWN ON A LIGHT-DARK CYCLE AND AFTER TRANSFER TO CONSTANT LIGHT

Abstract— The chlorophyll a fluorescence properties of Gonyaulax polyedra cells before and after transfer from a lightdark cycle (LD) to constant dim light (LL) were investigated. The latter display a faster fluorescence transient from the level ‘I’ (intermediary peak) to ‘D’ (dip) to ‘P’ (peak) than the former (3 s as compared to 10 s), and a different pattern of decline in fluorescence from ‘I’ to ‘D’ and from ‘P’ to the steady state level with no clearly separable second wave of slow fluorescence change, referred to as ‘s' (quasi steady state)→‘M’ (maximum) →‘T’ (terminal steady state). The above differences are constant features of cells in LD and LL, and are not dependent on the time of day. They are interpreted as evidence for a greater ratio of photosystem II/photosystem I activity in cells in LL. After an initial photoadaptive response following transfer from LD to LL, the cell absorbance at room temperature and fluorescence emission spectra at 77 K for cells in LL and LD are comparable. The major emission peak is at 685–688 nm (from an antenna Chl a 680, perhaps Chl a-c complex), but, unlike higher plants and other algae, the emission bands at 696–698 nm (from Chl a_II complex, Chl a 685, close to reaction center II) and 710–720 nm (from Chl a₁, complexes, Chl a 695, close to reaction center I) are very minor and could be observed only in the fluorescence emission difference spectra of LL minus LD cells and in the ratio spectra of DCMU-treated to non-treated cells. Comparison of emission spectra of cells in LL and LD suggested that, in LL, there is a slightly greater net excitation energy transfer from the light-harvesting peridinin-Chl a (Chl a 670) complex, fluorescing at 675 nm, to the other antenna chlorophyll a complex fluorescing at 685–688 nm, and from the Chl a., complex to the reaction center II. Comparison of excitation spectra of fluorescence of LL and LD cells, in the presence of DCMU, confirmed that cells in LL transfer energy more extensively from the peridinin-Chl a complex to other Chl a complexes than do cells in LD.     

EVENTS SURROUNDING THE EARLY DEVELOPMENT OF EUGLENA CHLOROPLASTS. 12. SPECTROSCOPIC EXAMINATION OF THE PROTOCHLOROPHYLL(IDE) PHOTOTRANSFORMATION IN INTACT CELLS*

Abstract— Absorption measurements in the 600–720 nm region of dark-grown cells of Euglena gracilis Klebs var. bacillaris Cori were made in vivo at room temperature using computer-assisted spectrophotometry. Dark-grown wild-type cells have a prominent absorption maximum at 634 nm due to protochlorophyll(ide) absorption. Upon illumination, the absorption at 634nm decreases and a peak appears at 674nm, representing the phototransformation of protochlorophyll(ide) to chlorophyll(ide). Using difference spectroscopy, the resynthesis in the dark of protochlorophyll(ide) by previously-illuminated wild-type dark-grown cells was found to begin at about 10min after illumination and reached completion by about 25 min, the amount of protochlorophyll(ide) resynthesized being equivalent to that of dark-grown cells. Resynthesis of protochlorophyll(ide) following a second illumination follows the same kinetics, indicating that protochlorophyll(ide) resynthesis is under tight regulation, possibly via feedback control. Cells of dark-grown wild-type and W₃BUL, a mutant lacking protochlorophyll(ide) contain a component absorbing at 658 nm which does not undergo phototransformation when examined by difference spectroscopy at room and liquid N₂ temperatures. Following the phototransformation of protochlorophyll(ide) 634 to chlorophyll(ide) 674, the chlorophyll(ide) 674 shifts to shorter wavelengths, ultimately to 671 nm. Possible relationships among the various spectroscopic forms of protochlorophyll(ide) and chlorophyll(ide) at room temperature and liquid N₂ temperature in Euglena and higher plants are presented. It is concluded that Euglena, unlike older, etiolated higher plants, contains only protochlorophyll(ide) 634, making it an excellent system in which to examine the phototransformation of this pigment species in the absence of other forms.     

EXCITON TRANSFER OUT OF OPEN PHOTOSYSTEM II REACTION CENTERS

Abstract A simple photochemical model of photosystem II consisting of antenna chlorophyll and a reaction center was used to examine the phenomenon of exciton detrapping, i.e. the transfer of excitation energy from open reaction centers back to the antenna. η, the ratio of the probability of detrapping when the reaction centers are all open, Ψ_t(o) to the probability when the centers are closed, Ψ_t(x) was used as a variable parameter to examine the various pathways of energy dissipation in a system in which P, the yield of photochemistry, and R, the ratio of the maximum to the minimum yields of fluorescence, were assumed to be known (e.g. R= 4.0 and P= 0.90). It is shown that η must fall within a range of values between 0 and R (1 –P) and that, for given values of R and P, Ψ_t(o) and the ratio of the rate constant for photochemistry at the reaction center, k_p, to the rate constant for energy transfer back to the antenna, k_t, can be determined for any assumed value of η. Even though detrapping occurs at open reaction centers, it is the magnitude of the yield of nonradiative decay at closed reaction centers, Ψ_a(x) which sets the upper limit on η. Equations for the overall yields of fluorescence and nonradiative decay in the antenna chlorophyll and of nonradiative decay at the reaction center chlorophyll, under conditions of both open and closed reaction centers, were derived in conventional probability terms and in terms of R, P and η. As η increases within its range of permissible values, energy dissipation in the antenna decreases and nonradiative decay at the reaction center increases. The determination of a specific value of η or of the ratio k_pk_t would require additional information such as the value of the maximum yield of fluorescence and the ratio of the rate constants for fluorescence and nonradiative decay in the antenna chlorophyll. The characteristics of a system in which there is no nonradiative decay in the reaction center (i.e. k_d= 0), in which case R (1 –P) = 1.0, were also examined. In this case the yield of detrapping has no influence on energy dissipation in the system. Finally, the question of heterogeneity in PSII was considered. It is suggested that Ψ_d(x) may be greater in PSII_β than in PSII_α so that the probability of detrapping could be greater in the PSII_α fraction.     

On the photodecomposition of chlorophyll in vitro. I. Reaction rates

Abstract— Chlorophyll solutions are irreversibly bleached by light in the presence of oxygen. The action spectra parallel the absorption spectra for both chlorophyll a and b. The reaction is of second order with a Q₁₀ of 1.26. The reaction rates for chlorophylls a and b are of the same order of magnitude. Depending upon the light source, the initial rate for chlorophyll a is slightly higher, by a factor of 1.15 to 1.30. The rate for pheophytin is lowe 3 by several orders of magnitude. No pheophytin has been detected in the reaction products of the irradiated chlorophyll solutions in the absence of water.     

A New Approach for the Photosynthetic Antenna–Reaction Center Complex with a Model Organized Around an s‐Triazine Linker

Two new artificial mimics of the photosynthetic antenna‐reaction center complex have been designed and synthesized (BDP‐H₂P‐C₆₀ and BDP‐ZnP‐C₆₀). The resulting electron‐donor/acceptor conjugates contain a porphyrin (either in its free‐base form (H₂P) or as Zn‐metalated complex (ZnP)), a boron dipyrrin (BDP), and a fulleropyrrolidine possessing, as substituent of the pyrrolidine nitrogen, an ethylene glycol chain terminating in an amino group C₆₀‐X‐NH₂ (X=spacer). In both cases, the three different components were connected by s‐triazine through stepwise substitution reactions of cyanuric chloride. In addition to the facile synthesis, the star‐type arrangement of the three photo‐ and redox‐active components around the central s‐triazine unit permits direct interaction between one another, in contrast to reported examples in which the three components are arranged in a linear fashion. The energy‐ and electron‐transfer properties of the resulting electron‐donor/acceptor conjugates were investigated by using UV/Vis absorption and emission spectroscopy, cyclic voltammetry, and femtosecond transient absorption spectroscopy. Comparison of the absorption spectra and cyclic voltammograms of BDP‐H₂P‐C₆₀ and BDP‐ZnP‐C₆₀ with those of BDP‐H₂P, BDP‐ZnP and BDP‐C₆₀, which were used as references, showed that the spectroscopic and electrochemical properties of the individual constituents are basically retained, although some appreciable shifts in terms of absorption indicate some interactions in the ground state. Fluorescence lifetime measurements and transient absorption experiments helped to elucidate the antenna function of BDP, which upon selective excitation undergoes a rapid and efficient energy transfer from BDP to H₂P or ZnP. This is then followed by an electron transfer to C₆₀, yielding the formation of the singlet charge‐separated states, namely BDP‐H₂P ^.+‐ C₆₀ ^.? and BDP‐ZnP ^.+‐ C₆₀ ^{. ?}. As such, the sequence of energy transfer and electron transfer in the present models mimics the events of natural photosynthesis.     

FAR-INFRARED SPECTRA OF LANGMUIR-BLODGETT FILMS OF CHLOROPHYLL a,CHLOROPHYLL b,PHEOPHYTIN a and THEIR ADDUCTS WITH WATER and DIOXANE*

Fourier transform infrared spectra in the low frequency region (500–150cm^?1) of Langmuir-Blodgett films of chlorophyll a (Chi a), chlorophyll b (Chi b) and pheophytin a have been studied. Correlations between spectral changes in monolayer and multilayers of Chi a and Chi b and their adducts with water and dioxane have been established. Spectroscopic evidence has indicated that, although there are no individual absorption bands that can be assigned to pure Mg-nitrogen and/or Mg-oxygen stretching or bending modes, there are several bands in the400–200 cm^?1 region of the spectra containing considerable contributions from metal-nitrogen and metal-oxygen vibrational modes. These specific vibrations exhibit marked intensity changes and shifts upon water and dioxane interaction. The different states of chlorophyll aggregation in Langmuir-Blodgett mono- and multilayers films resulted in noticeable changes in their far-IR spectra.     

A theoretical study of electronic excited states of photosynthetic reaction center in Rhodopseudomonas viridis

The electronic singlet vertical excited states of photosynthetic reaction center (PSRC) in Rhodopseudomonas (Rps.) viridis were investigated by ZINDO and INDO/S methods. The effects of the interactions of pigment-pigment and pigment-protein on the electronic excitations were examined. The calculation results showed that the interactions of pigment-pigment and pigment-protein play an important role in reasonably assigning the experimental absorption and circular dichroism (CD) spectra of PSRC in Rps. virids. By comparing the theoretically computed excited states with the experimental absorption and CD spectra, satisfactory assignments of the experimental spectroscopic peaks were achieved.     

ENERGY TRANSFER IN SELF-ASSEMBLED CHLOROPHYLL a SYSTEMS *

Abstract Computer deconvolution of the optical absorption spectra has been used to follow the formation and transformation of various chlorophyll species formed by lowering the temperature in solutions of toluene or methylcyclohexane containing a hydrogen-bonding nucleophile such as ethanol. At cryogenic temperatures the self-assembled chlorophyll a-ethanol species has optical properties similar to those of special pair photoreaction center chlorophyll. Analysis of the absorption and fluorescence behavior of the self-assembled species suggests that chlorophyll-chlorophyll species are also formed in the self-assembly process. Fluorescence lifetime measurements indicate that the pathways for dissipation of excitation energy in these multicomponent systems are complex. Selective optical excitation at wavelengths corresponding to absorption of monomer, oligomer, etc. chlorophyll a species has been used to demonstrate the heterogeneous nature of these self-assembled systems.     

Absorption and Fluorescence Spectral Database of Chlorophylls and Analogues

Absorption spectra and fluorescence spectra are essential for use across the photosciences, yet such spectra along with the all‐important values for molar absorption coefficient (ε) and fluorescence quantum yield (Φ_f) often are found with great difficulty. Here, a literature survey concerning the vital class of chlorophyll compounds has led to identification of spectra for 150 members. Spectra in print form have been digitized (with baseline corrections) and assembled into a database along with literature references, solvent identity and values for ε and Φ_f (where available). The database encompasses photosynthetic tetrapyrroles wherein the chromophore is a porphyrin (e.g. chlorophyll c₁, protochlorophyll a), chlorin (e.g. chlorophyll a, bacteriochlorophyll c) or bacteriochlorin (e.g. bacteriochlorophyll a). Altogether, the database contains 305 absorption spectra (from 19 porphyrins, 109 chlorins and 22 bacteriochlorins) and 72 fluorescence spectra (from 10 porphyrins, 30 chlorins and 4 bacteriochlorins). The spectral database should facilitate comparisons and quantitative calculations. All spectra are available in print form in the Supporting Information. The entire database in digital form is available with the PhotochemCAD program for free downloading and further use at http://www.photochemcad.com .     

STRUCTURE OF PHOTOSYSTEM I AND PHOTOSYSTEM II OF PLANT CHLOROPLASTS*,†,§

Abstract— The action of Triton X-100 upon photosynthetic membranes which are devoid of carotenoids produces a small Photosystem I particle (HP700 particle) which is active in N ADP photoreduction and has a [Chl]/[P700] ratio of 30. The properties of the HP700 particle indicate that it is a reaction center complex which is served by an accessory complex containing the additional light-harvesting chlorophyll of Photosystem I as well as the cytochromes and plastoquinone. When Photosystem II particles obtained by the action of Triton X-100 are further washed with a solution 0.5 M in sucrose and 0.05 M in Tris buffer (pH 8.0), chlorophyll-containing material is released. After centrifugation, the supernatant contains about 1 per cent of the chlorophyll and contains three types of particles which can be separated by sucrose density gradient centrifugation. One of these particles, designated TSF-2b, has the same pigment composition as the original Photosystem II fragment, contains cytochrome 559, and shows Photosystem II activity (DCMU-sensitive diphenylcarbazide-supported photoreduction of 2,6-dichlorophenolindophenol). The other two particles (TSF-2a and TSF-2a′) have a [Chl a]/[Chl b] ratio of 8, have a low concentration of xanthophylls, and show a [Chl]/[Cyt 5591 ratio of about 20. Only the TSF-2a particle is active in the Photosystem II reaction described above. On the basis of these data, it is proposed that the Photosystem II unit consists of a reaction center complex which contains Chl a, Cyt 559, and an acceptor for the photochemical reaction. The reaction center complex would be served by an accessory complex which contains the light-harvesting pigments, Chl a. Chi b, and xanthophyils.     

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

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

STUDIES OF CHLOROPHYLL-CHLOROPHYLL AND CHLOROPHYLL-LIGAND INTERACTIONS BY VISIBLE ABSORPTION AND INFRARED SPECTROSCOPY AT LOW TEMPERATURES

Abstract— A comparison of the visible absorption and infrared spectra of various chlorophyll-chlorophyll (Chl) and Chi-nucleophile aggregates at room temperature and at low temperatures has been made. The IR data provide structural information indispensable for the interpretation of the visible spectra. As a necessary preliminary, it is shown that Chl a solutions in nonpolar solvents can be prepared by appropriate drying techniques that contain at a conservative estimate ≤ 3 mol % of water (i.e. Chl a/H₂O > 30:1). Very dry solutions of Chl a or Pyrochl a(≥ 10 mM) in toluene or methylcyclohexane-isopentane solution show only slight changes in visible spectra on cooling to 77 K. From IR, additional Chl-Chl aggregation occurs on cooling in methylcyclohexane-isopentane but not to a significant extent in toluene. Dilute (10 μM) solutions of Chl a or Pyrochl a in nonpolar solvents form a new absorption peak near 700 nm at low temperatures, which we attribute to traces of water in the solvent or other residual nucleophiles not removed during the Chl purification. Addition of stoichiometric amounts of water increases the size of the ?700 nm peak even in dilute Chl solutions. Chlorophyll a, Pyrochl a, but not pheophytin a are shown to interact with nucleophiles of the general type RXH (where R= H or alkyl, and X = O, N, or S). Such nucleophiles can coordinate to the Mg atom of one Chl molecule by lone pairs on O, N, or S, and hydrogen bond to oxygen donor functions in another Chl molecule. A ?0.1 M solution of Chl a or Pyrochl a in toluene containing 1.5 equivalents of ethanol is converted almost entirely to a species absorbing at ?700 nm at 77 K. Infrared spectroscopy shows conclusively that it is the keto C=O function that is involved in the cross-linking by hydrogen bonding, a conclusion supported by the observation that Pyrochl a forms a very similar red-shifted species at low temperatures, despite the absence of a carbomethoxy C=O function. n-Butylamine and ethanethiol interact in much the same way as does ethanol to form species red shifted to ?700 nm. A variety of possible structures for the low temperature forms is discussed, and the use of these red shifted species as paradigms for photoreaction center Chl is described.