Chlorophyll and peptide compositions in the two photosystems of marine green algae.

The molar ratios of chlorophyll a to b in the thalli of marine green algae were between 1.5 and 2.2, being appreciably lower than the ratio between 2.8 and 3.4 found for the leaves of higher plants and the cells of fresh-water green algae. The ratio of chlorophylls to P-700 in these marine algae was also lower than that in higher plants. The a/b ratios in the pigment proteins of Photosystems 1 and 2 separated by polyacrylamide-gel electrophoresis from sodium dodecyl sulfate-solubilized chloroplasts of four species of marine green algae, Bryopsis maxima, Cheatomorpha spiralis, Enteromorpha compressa and Ulva conglobata, were approximately 5 and 1, which are considerably smaller than the ratios, 7 and 2, respectively, found for the pigment proteins of the two photosystems of higher plants separated by the same technique. The chloroplasts of Bryopsis maxima and Cheatomorpha spiralis lacked two of the peptides associated with Photosystem II, which are present in the chloroplasts of Spinacia oleracea and Taraxacum officinale.     

Chlorophyll-protein complexes of a Codium species,including a light-harvesting siphonaxanthin-Chlorophylla ab-protein complex,an evolutionary relic of some Chlorophyta

Eight chlorophyll-protein complexes were isolated from thylakoid membranes of a Codium species, a marine green alga, by mild SDS-polyacrylamide gel electrophoresis. CP 1a¹, CP 1a², CP 1a³ and CP 1a⁴ were partially dissociated Photosystem (PS) I complexes, which in addition to the core reaction centre complex, CP 1, possessed PS I light-harvesting complexes containing chlorophyll (Chl) a, Chl b and siphonaxanthin. LHCP¹ and LHCP³ are orange-brown green chlorophyll $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 0.66) that contain siphonaxanthin and its esterified form, siphonein. CP a and CP 1, the core reaction centre complexes of PS II and PS I, respectively, had similar spectral properties to those isolated from other algae or higher plants. These P-680- or P-700-Chl a-proteins are universally distributed among algae and terrestrial plants; they appear to be highly conserved and have undergone little evolutionary adaptation. Siphonaxanthin and siphonein which are present in the Codium light-harvesting complexes of PS II and PS I are responsible for enhanced absorption in the green region (518 and 538 nm). Efficient energy transfer from both xanthophylls and Chl b to only Chl a in Codium light-harvesting complexes, which have identical fluorescence emission spectra at 77 K to those of the lutein-Chl $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 1.2) of most green algae and all higher plants, proved that the molecular arrangement of these light-harvesting pigments was maintained in the isolated Codium complexes. The siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ allow enhanced absorption of blue-green and green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats. Since there is a variable distribution of lutein, siphonaxanthin and siphonein in marine green algae and siphonaxanthin is found in very ancient algae, these novel siphonein-siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ may be ancient light-harvesting complexes which were evolved in deep water algae.     

Characterization of Chlorophyll–protein Complexes Isolated from Two Marine Green Algae, Bryopsis maxima and Ulva pertusa, Growing in the Intertidal Zone

Three Chl–protein complexes were isolated from thylakoid membranes of Bryopsis maxima and Ulva pertusa, marine green algae that inhabit the intertidal zone of the Pacific Ocean off the eastern coast of Japan by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. The slowest-moving fractions showed low Chl a/b and Chl/P-700 ratios, indicating that this fraction corresponds to complexes in PS I, which is large in both algae. The intermediate and fastest-moving fractions showed the traits of PS II complexes, with some associated Chl a/b–protein complexes and LHC II, respectively. The spectral properties of the separated Chl–proteins were also determined. The absorption spectra showed a shallow shoulder at 540 nm derived from siphonaxanthin in Bryopsis maxima, but not in Ulva pertusa. The 77 K emission spectra showed a single peak in Bryopsis maxima and two peaks in Ulva pertusa. Besides the excitation spectra indicated that the excitation energy transfer to the PS I complexes differed quite a lot higher plants. This suggested that the mechanisms of energy transfer in both of these algae differ from those of higher plants. Considering the light environment of this coastal area, the large size of the antennae of PS I complexes implies that the antennae are arranged so as to balance light absorption between the two photosystems. In addition, we discuss the relationships among the photosystem stoichiometry, the energy transfer, and the distribution between the two photosystems.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

Macromolecular organization of chlorophyll a in aggregated chlorophyll ab protein complex as shown by circular dichroism at room and cryogenic temperatures

This report concerns the large circular dichroic (CD) signal of intact chloroplasts of higher plants. The CD spectra of chloroplasts are compared with the aggregated form of the light-harvesting chlorophyll $\text{a}\text{b}$ complex at 25°C and ?250°C. The light-harvesting chlorophyll aggregate has a CD of magnitude equal to or greater than chloroplasts, but of opposite sign, and it is not related to the CD of the unaggregated form, and hence its arrangement is an artefact compared to the arrangement in the chloroplast. We suggest that this preparation, which has pseudo-lamellar structure, is a clear example of a large CD signal being generated by macromolecular association. The asymmetry of organization in the chloroplast has an opposite sense to that of the aggregate, but affects only chlorophyll a, not chlorophyll b.     

Antenna replacement in the evolutionary origin of chloroplasts

The endosymbiotic origin of chloroplasts from unicellular cyanobacteria is presently beyond doubt. Oxygenic photosynthesis is based on coordinated action of the two photosystems (PS), PS I and PS II, cooperating with several variants of the pigment antenna. In cyanobacteria, red algae, and glaucophytes, phycobilisomes (PBS) act as antennae, while in terrestrial plants, as well as in most macro- and microalgae, antennae are formed by chlorophyll a/b- and chlorophyll a/c-containing proteins. Advantages and disadvantages of the PBS antenna compared to other light-harvesting complexes form the basis for adaptive variations of the antenna in the course of development of eukaryotic photosynthesis. During the evolution of the “green” and “chromophyte” lineages of the chloroplasts, PBS, in spite of their optimal features of light absorption, were replaced by chlorophyll a/b- and chlorophyll a/c-containing light-harvesting complexes. Development of the cell wall associated with the limitation of motility and tissue formation in photosynthetic eukaryotes were the factors responsible for the antenna shift. The subsequent redistribution of cell resources in favor of cellulose biosynthesis required for increased CO₂ consumption, higher PS II levels, and greater number and density of the thylakoids in the chloroplasts, was incompatible with the energy-consuming and overly large PBS antenna.     

Spectral and functional studies on siphonaxanthin-type light-harvesting complex of photosystem II from Bryopsis corticulans

Carotenoids with conjugated carbonyl groups possess special photophysical properties which have been studied in some water-soluble light-harvesting proteins (Polívka and Sundström, Chem Rev 104:2021–2071, 2004). However, siphonaxanthin-type light-harvesting complexes of photosystem II (LHCII) in siphonous green alga have received fewer studies. In the present study, we determined sequences of genes for several Bryopsis corticulans Lhcbm proteins, which showed that they belong to the group of major LHCII and diverged early from green algae and higher plants. Analysis of pigment composition indicated that this siphonaxanthin-type LHCII contained in total 3 siphonaxanthin and siphonein but no lutein and violaxanthin. In addition, 2 chlorophylls a in higher plant LHCII were replaced by chlorophyll b. These changes led to an increased absorption in green and blue-green light region compared with higher plant LHCII. The binding sites for chlorophylls, siphonaxanthin, and siphonein were suggested based on the structural comparison with that of higher plant LHCII. All of the ligands for the chlorophylls were completely conserved, suggesting that the two chlorophylls b were replaced by chlorophyll a without changing their binding sites in higher plant LHCII. Comparisons of the absorption spectra of isolated siphonaxanthin and siphonein in different organic solutions and the effect of heat treatment suggested that these pigments existed in a low hydrophobic protein environment, leading to an enhancement of light harvesting in the green light region. This low hydrophobic protein environment was maintained by the presence of more serine and threonine residues in B. corticulans LHCII. Finally, esterization of siphonein may also contribute to the enhanced harvesting of green light.     

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ～1.9, ～1.5, and 1.1–1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments — carotenoids, Chl b, and phycobilins — were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.     

Resolution of the light-harvesting chlorophyll ab-protein of Vicia faba chloroplasts into two different chlorophyll-protein complexes

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll $\text{a}\text{b}$ ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll $\text{a}\text{b}$ ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.     

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes,thylakoids and chlorophyll-protein complexes

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845–854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to ?196°C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelength positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.     

Chlorophyllide b

Chlorophylls a,b and chlorophyllides a,b were isolated from pea chloroplasts as pheophorbides a,b following the administration of $\text{[}{}^{14}\text{C]δ-aminolevulinic}$ acid. Relative pool sizes suggest that chlorophyllide b precedes chlorophyll b and does not arise from the latter by the action of chlorophyllase.     

Isolation of the light-harvesting chlorophyll a/b--protein complex from thylakoid membranes of barley by adsorption chromatography on controlled-pore glass.

The light-harvesting chlorophyll $\text{a}\text{b}$-protein complex has been isolated from barley thylakoids by a rapid, single-step procedure involving adsorption chromatography on controlled-pore glass columns. The Triton X-100-solubilized complex contains a polypeptide of apparent molecular weight, 26,000; the 0.25% Triton X-100 light-harvesting chlorophyll $\text{a}\text{b}$-protein has spectral characteristics consistent with its assumed in vivo state. On the same column free chlorophyll and carotenoids have been separated from chlorophyll-protein complex 1, but this complex contained many polypeptides other than those associated with chlorophyll. This method is potentially suitable for the isolation of other thylakoid membrane proteins. It may also be generally applicable for fractionation of intrinsic membrane proteins from other sources and for separation of mixed Triton X-100-lipid micelles.     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

A comparison of plants regenerated from a variegated <Emphasis Type="Italic">Epipremnum aureum</Emphasis>

In order to study chloroplast biogenesis, we chose natural variegated Epipremnum aureum (golden pothos) and regenerated pale yellow, variegated and green plants from all three types of tissue explants. The percentage of three types of regenerated shoots from three different explants was very close. Regenerated plants have been maintained for a year and show no sign of a colour switch. By comparing their protein profiles, two major differences between pale yellow and green plants were observed at the 15 and 40 to 50 kDa proteins. Moreover, pale yellow plants had unexpected high molecular mass proteins (greater than 60 kDa). Both variegated and green plants had more chlorophyll (Chl) a than Chl b, the ratios were about 1.46 and 1.93, respectively. In contrast, the pale yellow plants not only had less total Chl, but also the reduction of Chl a was much greater than Chl b, resulting in a higher content of Chl b than Chl a. Microscopic analysis revealed that pale yellow plants contained predominantly undeveloped chloroplasts with low Chl contents, even though their mesophyll cells were similar to green and variegated plants. PCR amplification of chloroplast DNA with 14 universal chloroplast primers did not reveal any difference among these regenerated plants.     

Linear dichroism of microalgae,developing thylakoids and isolated pigment-protein complexes in stretched poly (vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields.Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b (648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl $\text{a}\text{b}$ ratio of approx. 6 and the LD spectrum was positive with a maximum at 690–694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack Chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

The P700-chlorophyll a-protein. Isolation and some characteristics of the complex in higher plants

A P700-chlorophyll a-protein complex has been purified from several higher plants by hydroxylapatite chromatography of Triton X-100-dissociated chloroplast membranes. The isolated material exhibits a red wavelength maximum at 677 nm, major spectral forms of chlorophyll a at 662, 669, 677, and 686 nm, a chlorophyll/P700 ratio of $\text{40–45}\text{1}$, and contains only chlorophyll a and β-carotene of the photosynthetic pigments present in the chloroplast. The spectral characteristics and composition of the higher plant material are homologous to those of the P700-chlorophyll a-protein previously isolated from blue-green algae; however, unlike the blue-green algal component, cytochromes f and b₆ are associated with the higher plant material. Evidence is presented that a chlorophyll a-protein termed “Complex I” which can be isolated from sodium dodecyl sulfate extracts of chloroplast membranes is a spectrally altered form of the eucaryotic P700-chlorophyll a-protein. The isolation procedure described in this paper is a more rapid technique for obtaining the heart of photosystem I than presently exists; furthermore, the P700 photooxidation and reduction kinetics in the fraction are improved over those in other isolated components showing the same enrichment of P700. It appears very probable that the heart of photosystem I is organized in the same manner in all chlorophyll a-containing organisms.     

Effects of cations upon chloroplast membrane subunit interactions and excitation energy distribution

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under “low salt” conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll · protein complex (chlorophyll $\text{a}\text{b}$ ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll $\text{a}\text{b}$ protein and which acts as a light-harvesting antenna primarily for Photosystem II.Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll b and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment · protein, the combined complexes pellet as a “heavy” membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a “light” submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation.Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment · protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.     

Properties of thylakoids and thylakoid particles derived from structurally different chloroplasts

Structurally and functionally different tobacco chloroplasts were subjected to digitonin treatment and subsequent fractional centrifugation. The light-harvesting $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b-}\text{protein}$ complex was found to be enriched in the most dense fraction regardless of the presence of grana in the original preparation. It is suggested that isolated thylakoid membranes and fragments thereof which contain sufficient light-harvesting protein may, under appropriate ionic conditions, form aggregates even when they originate from unstacked thylakoid systems. Comparative studies of fluorescence properties and polypeptide composition of the thylakoids suggest that the light-harvesting protein does not contribute significantly to the fluorescence spectrum of isolated chloroplasts as long as this protein is intimately associated with the Photosystem II (PS II) pigment-protein complex responsible for the 685 nm emission. While the PS II-deficient mutant chloroplasts of the variegated tobacco variety NC 95 lacked both the 685 nm fluorescence component and two or three PS II proteins, one of these proteins was found to be very prominent in our chlorophyll b-deficient mutant thylakoids which also displayed an intense 685 nm fluorescence peak. This correlation supports the contention that a 45 kdalton polypeptide is an apoprotein of pigments associated with the PS II reaction center.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.