PROTEIN COMPOSITION OF SPINACH CHLOROPLASTS AND THEIR PHOTOSYSTEM I AND PHOTOSYSTEM II SUBFRAGMENTS*

Abstract— The proteins of spinach chloroplasts and their subfragments containing photosystem I and photosystem II, obtained by Triton X-100 treatment or French-pressure rupture, were separated by sodium dodecyl sulfate (SDS)-acrylamide electrophoresis at pH 7·0 in phosphate buffer. The individual protein bands were identified where possible by comparing them with known, isolated and characterized proteins from chloroplasts, and their molecular weights were determined. The protein composition of the chloroplast fragments were correlated to the functional properties of these fragments. Distinct patterns were obtained for photosystem I and photosystem II particles. The major protein of photosystem II is expressed in the 23 kilodalton range and photosystem I proteins seem to be clustered mainly in the 50–70 kilodalton range.     

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.     

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X-100. CP1a has a Chl a / b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X-100 PS I complex (Chl a / b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a / b -proteins of the main light-harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X-100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a / b -proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.     

ON THE INTERACTION OF PHOTOSYSTEMS I AND II IN ALGAL CELLS*,†

Abstract— Steady-state relaxation spectrophotometry was applied to intact algal cells. The results indicated System I operates in a cyclic manner, only indirectly affected by the presence or absence of oxygen evolution. This action in whole cells is distinct from that occurring in chloroplasts, where System I appears tightly coupled to System II. No evidence was found for the oxidation of cytochrome by P700. Fluorescence emission measurements on intact cells emphasized the role of membrane permeability agents in controlling the emission yield. Large changes in the yield caused by Systems I and II could be observed in the presence of these agents. In sum, the results proved complex and were difficult to resolve in any simple scheme but emphasized the possible role of protons (or membrane potential) in controlling fluorescence yield.     

BIPHASIC ENERGY CONVERSION KINETICS AND ABSORBANCE DIFFERENCE SPECTRA OF PHOTOSYSTEM II OF CHLOROPLASTS. EVIDENCE FOR TWO DIFFERENT PHOTOSYSTEM II REACTION CENTERS

Abstract— The continuous illumination induced kinetics of photochemical energy conversion at system II have been measured with isolated and 3-(3, 4-dichlorophenyl)-l, l-dimethylurea (DCMU) poisoned chloroplasts by means of absorbance difference spectroscopy in the UV and by the area growth over the fluorescence induction curve at room temperature. An optimal set of conditions was found in order to isolate absorbance changes caused by the reduction of the primary electron acceptor Q of PS II by suppressing other electron transfer processes. The light induced kinetics of Q- accumulation in the absorbance change measurements were found to be biphasic and strictly correlated with the kinetics of the area growth measured under the same conditions. From the resolution of the biphasic kinetics at different wavelengths in the UV region of the spectrum, it was found that both kinetic components in the system II photochemistry involve the reduction of a plastoquinone molecule to its plastosemiquinone anion. From the two kinetic components one was fast and non-exponential and the other relatively slow with an exponential time course. The initial rate difference in the kinetics of the two components was by a factor of approximately 3. A difference by a factor of about three was also found in the flash saturation curves of the two kinetic components.
The results are explained by the hypothesis that in higher plant chloroplasts there are system II reaction centers embedded in a large pigment matrix with statistical energy transfer, and system II reaction centers embedded in separate, in terms of excitation energy transfer, units. The effective absorption cross section per reaction center for the centers in the statistical pigment bed is approximately 3 times larger than that of the reaction centers in the separate system II units. The two types of system II reaction centers have different yields of excitation trapping and charge stabilization properties.     

THE PRIMARY REACTION OF PLANT PHOTOSYSTEM II

Abstract. …Research during the past five years has resulted in considerable advances in our understanding of the primary reaction (the initial light-induced charge separation) of plant Photosystem II (the reaction responsible for the oxidation of H₂O to O₂). The primary reaction appears to involve the photooxidation of a specialized chlorophyll a and the concomitant photoreduction of a specialized plastoquinone. Evidence for this formulation of the Photosystem II reaction center and a discussion of the kinetic and oxidation-reduction properties of the reactants are reviewed.     

ALTERNATIVE MEASURES OF PHOTOSYSTEM II ELECTRON TRANSFER INHIBITION IN ANTHRAQUINONE-TREATED CHLOROPLASTS

We have previously used chlorophyll fluorescence measurements at Fmax conditions (i.e. with Photosystem II electron acceptor QA reduced) to monitor the action of 9,10-anthraquinones on photosynthetic electron transport in plant chloroplasts. The present investigation employs two additional techniques to characterize the extent of electron transport inhibition induced by the addition of substituted anthraquinones to the suspending medium of spinach chloroplasts. Results are presented for spectrophotometric assays of the rate of electron transfer to an exogenous electron acceptor, 2,6-dichloroindophenol (DCIP) and for electrochemical determinations of the rate of oxygen evolution in anthraquinone-treated chloroplasts. In general, amino-substituted anthraquinones are ineffective inhibitors, maintaining electron transfer rates to DCIP at levels ranging from 50 to 90% of normal rates and yielding rates of O2 evolution averaging at 70% of the rate in untreated chloroplasts. In contrast, hydroxy-substituted anthraquinones efficiently block Photosystem II electron transport, resulting in low rates of DCIP photoreduction ranging from 0 to 20% of normal values and reducing O2 evolution rates to an average of 30% of the rate observed for untreated chloroplasts. Relative rates of DCIP photoreduction for anthraquinone-treated chloroplasts show a strong linear correlation with the reported relative Fmax chlorophyll fluorescence intensities. Relative O2 evolution rates are observed to correlate with the Stern-Volmer fluorescence quenching parameter Ksv. We suggest that slight differences in the extent of inhibitory activity of an anthraquinone as measured by the three techniques are consistent with certain known Photosystem II heterogeneities. The similarities in relative rankings of inhibitory effects for the 9, 10-anthraquinones, however, demonstrate that the three techniques employed (measurements of Fmax chlorophyll fluorescence, DCIP photoreduction rates, and O2 evolution rates) are alternative assays of anthraquinone-induced Photosystem II electron transport inhibition.     

EFFECTS OF PHOTOSYSTEM II INHIBITORS ON ELECTRON PARAMAGNETIC RESONANCE SIGNAL II OF SPINACH CHLOROPLASTS

Abstract— In isolated spinach chloroplasts the light-induced electron paramagnetic resonance signal (signal II) associated with the oxygen evolving photosystem (photosystem II) decays slowly and incompletely in the dark. Tris-washing, hydroxylamine, or carbonylcyanide m -chlorophenylhydrazone (CCCP) enhance the decay of signal II, which can still be induced by red (645 nm) but not by far-red (735 nm) radiation. Although 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) alone has no effect on signal II, it blocks the induction of signal II in the presence of hydroxylamine or CCCP. These data suggest that signal II is an indicator of an oxidized intermediate on the water-splitting side of photosystem II.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE DYNAMICS OF PHOTOSYSTEM I IN GREEN-PLANT PHOTOSYNTHESIS—II. INTACT AND BROKEN SPINACH CHLOROPLASTS*

Abstract —The transient oxidation and subsequent reduction of P700⁺ in spinach chloroplasts has been monitored by flash photolysis-electron spin resonance spectroscopy in the presence of various donors and acceptors. In general, the results obtained correlate well with results on Photosystem I subchloroplast particles, with two major differences. For Type A and B intact chloroplasts in the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU), the electron acceptor methyl viologen has no effect on the decay kinetics. This phenomenon is interpreted in terms of a functioning cyclic electron flow path around Photosystem I. Also, the photoresponse of Signal I depends on the length of the photolyzing flash. This is interpreted in terms of the existence of a primary electron donor to P700⁺ with a transfer time of ? 10 μs.     

QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron-transport were estimated from the concentration of PSII reaction-centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA₃₂₀] and green [ΔA_540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron-transport were measured under the same light-limiting conditions using 2,5-dimethylbenzoquinone or 2,5-dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSII_β) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady-state electron transport in isolated chloroplasts and caused underestimation of PSII electron-transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash-yield measurements. It is implied that quantitation of PSII by repetitive flash-yield methods requires assessment of the slow turnover of electrons by PSII_β and, in the presence of added quinones, assessment of the PSII quantum yield.     

NEW PHOTOSYSTEM I ELECTRON ACCEPTORS: IMPROVEMENT OF HYDROGEN PHOTOPRODUCTION BY CHLOROPLASTS *

Abstract Four novel electron carriers (two zwitterionic bipyridyls, dicarboxyl colbalticinium and sodium metatungstate), which are negatively charged in their reduced form, have been tested as photo-system I acceptors and as mediators of H₂ evolution. Measurements of O₂ uptake, anaerobic photoreduction rates and stationary concentrations of reduced species under continuous illumination indicate that Coulombic interactions control the electron transfer between the photosynthetic membrane and the mediators. Both rates of forward transfer and back reaction (electron cycling) seem to depend on the charge of the electron carrier. The low concentration of anionic species in the diffuse layer associated with the membrane could explain our results. Hydrogen evolution rates obtained with these four mediators used as electron relays between the photosynthetic membrane and colloidal platinum catalyst are higher than with methylviologen. This improvement of the conversion efficiency parallels the high steady state accumulation of reduced carriers favoured by their negative charge. It is also shown that these synthetic mediators, except metatungstate, are able to evolve hydrogen with an hydrogenase isolated from Desulfovibrio desulfuricans.     

THE MECHANISM OF DELAYED LIGHT PRODUCTION BY PHOTOSYNTHETIC ORGANISMS AND A NEW EFFECT OF ELECTRIC FIELDS ON CHLOROPLASTS*,‡

Abstract— Green plants, after illumination, emit light at times far too long to be fluorescence. This delayed light is closely connected with the process of photosynthesis and seems to be one of the few ways of studying the first steps in that process. In this paper we argue that there are at least 3 or maybe 4 mechanisms producing delayed light. (1) The delayed light in the range of 1–100 msec seems to come from the recombination of electrons and holes. The photosynthetic unit must absorb 2 quanta for this process. (2) At longer times the delayed light can come from thermal fluctuations lifting an electron from the level of ferredoxin to that of chlorophyll. The unit need only absorb 1 light quantum for this kind of delayed light. (3) Similarly, a part of the long-time delayed light comes from the untrapping of holes. (4) A part of the delayed light emitted at times longer than a few minutes seems to involve molecular oxygen. Finally, we shall describe a new phenomenon involving the effect of electric fields on chloroplasts, that we feel will be helpful in understanding the untrapping mechanisms of delayed light production.     

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

INHIBITION OF ELECTRON TRANSPORT IN CHLOROPLASTS BY CHAOTROPIC AGENTS AND THE USE OF MANGANESE AS AN ELECTRON DONOR TO PHOTOSYSTEM II*

Abstract— Incubating spinach chloroplasts with various chaotropic agents results in inhibition of photosynthetic electron transport between water and Photosystem II similar to the inhibition caused by washing chloroplasts with a high concentration of Tris buffer. Partial restoration of NADP photoreduction and fluorescence of variable yield is achieved by adding hydroquinone or Mn²⁺, either of which donates electrons to Photosystem II in the inhibited chloroplasts. The inhibitory treatments cause the release of Mn from its bound state in the chloroplast, thus allowing the measurement of the ESR signal of Mn²⁺. The ESR measurement is used to follow the photooxidation of Mn²⁺ as it donates electrons to photosystem II.     

THE STRUCTURE OF CHLOROPHYLL RC I, A CHROMOPHORE OF THE REACTION CENTER OF PHOTOSYSTEM I

Abstract— Chlorophyll RC I is a particular chlorophyll of photosystem I common to all organisms with oxygenic photosynthesis. Its structure could be revealed by'H-NMR, FTIR, neutron activation analysis, complemented by plasma desorption mass spectrometry data. It has been identified as 13'-hydroxy-20-chloro-Chl a . Two stereoisomers of Chl RC I have also been isolated and identified. Evidence is presented that chlorination of the pigment does not occur during extraction and that artefacts due to impurities are ruled out.     

PICOSECOND TRANSIENT BEHAVIOR OF REACTION-CENTER PARTICLES OF PHOTOSYSTEM I ISOLATED FROM SPINACH CHLOROPLASTS. ENERGY AND ELECTRON TRANSFER UPON SINGLE AND MULTIPLE PHOTON EXCITATION

Abstract— Both [15-¹³C] and [14-¹³C] all-trans-retinals were synthesized. Bacteriorhodopsin containing [14-¹³C]retinal as a chromophore, when solubilized with octyl-β-D-glucoside, showed characteristic resonances at 125 and 118 ppm from tetramethyl silane. The former was assigned to the signal from free retinal and the latter from protonated Sehiff base. When the bacteriorhodopsin was denatured in sodium dodecyl sulfate, the signal at 118 ppm disappeared, while the signal at 125 ppm rather increased.
In the case of bacteriorhodopsin containing [15-¹³C]retinal, when solubilized with Triton X-100, a characteristic resonance at 169 ppm was distinguishable as a shoulder peak superimposed on the broad signal of carbonyl carbons and it was assigned to the signal from the protonated Sehiff base. The other signal observed at 191 ppm was from free retinal.
These results suggested that the Sehiff base of bacteriorhodopsin is protonated in the dark.     

BIOSYNTHESIS OF PTERIDINES IN Neurospora crassa,Phycomyces blakesleeanus AND Euglena gracilis: DETECTION AND CHARACTERIZATION OF BIOSYNTHETIC ENZYMES*,†

Abstract— Occurrence, biosynthesis and some functions of tetrahydrobiopterin (H₄biopterin) in animals are well known. The biochemistry of H₄biopterin in other organisms than animals was hitherto not widely investigated. Recently H₄biopterin was found in the phytoflagellate Euglena gracilis, in the zygomycete Phycomyces blakesleeanus and in the ascomycete Neurospora crassa. In Euglena, Neurospora and Phycomyces the enzymatic activities of GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are detectable and the biosynthesis follows the same steps as were shown for animals. The biosynthetic enzymes, however, show a much lower sensitivity to those inhibitors that act on vertebrate enzymes. 2,4-Diamino-6-hydroxypyrimidine as inhibitor of GTP cyclohydrolase I and N-acetylserotonin or N-methoxyacetylserotonin as inhibitors of sepiapterin reductase can decrease pteridine biosynthesis significantly, in vitro and in vivo. The apparent K_mvalues are in general higher when compared with the respective animal enzymes. In Neurospora, the conversion of GTP to dihydroneopterin triphosphate was closely associated with subsequent production of 6-hydroxymethyl-7, 8-dihydropterin due to the high activity of dihydroneopterin aldolase, different from all other tested organisms. Investigations involving inhibition of pteridine synthesis might be a useful tool for evaluating the hypothesis that pteridines in fungi and plants are co-chromophores of various blue light-dependent, flavin-containing phototrcptors.     

BIMANES—26. AN ELECTRON TRANSFER REACTION BETWEEN PHOTOSYSTEM I1 AND MONOBROMOBIMANE INDUCES STATIC CHLOROPHYLL a QUENCHING IN SPINACH CHLOROPLASTS

Abstract— Monobromobimane in chloroplasts lowers both the quantum yield of system II photochemistry and the yield of chlorophyll a fluorescence. Illumination of the chloroplasts in the presence of monohromobimane is an absolute prerequisite to the manifestation of this phenomenon, which proceeds via the Photosystem II intermediate, the semiquinone radical anion, Q_A-. The latter transfers an electron to monobromobimane to yield an anion radical (mBBr^·), which may either lose bromide ion to yield a reactive radical (mB^·), or acquire a proton and undergo further reduction, eventually forming syn-(methyl, methyl) bimane. In turn, mB reacts with the protein of the light-harvesting complex, to form a product which acts as static excitation energy quencher in the chlorophyll pigment bed of photosystem 11. Polarographic reduction of monobromobimane shows an adsorption wave at O V and two reduction waves. Prolonged reduction in water at -0.5 V yields syn-(methyl, methyl) bimane (which is further reduced at more negative potentials) and bromide ion. Thus, both electrochemical and chloroplast-induced reduction produce syn-(methyl, methyl) bimane. Monobromobimane may then serve as a Photosystem II activated probe in elucidating the conformation of intrinsic thylakoid membrane polypeptides.     

Monomere und polymere Dimethylaminothioquadratato‐Komplexe: Die Kristallstrukturen von Nickel(II)‐, Cobalt(II)‐, Silber(I)‐, Platin(II)‐, Gold(I)‐, Quecksilber(II)‐ und Blei(II)‐Dimethylaminothioquadrataten

Monomeric and Polymeric Dimethylaminothiosquarato Complexes: The Crystal Structures of Nickel(II), Cobalt(II), Silver(I), Platinum(II), Gold(I), Mercury(II) and Lead(II) Dimethylaminothiosquarates The ligand 2‐dimethylamino‐3, 4‐dioxo‐cyclobut‐1‐en‐thiolate, Me₂N‐C₄O₂S⁻ (L) forms neutral and anionic complexes with nickel(II), cobalt(II)‐, silver(I)‐, platinum(II)‐, gold(I)‐, mercury(II)‐ and lead(II). According to the crystal structures of seven complexes the ligand is O, S‐chelating in [Ni(L)₂(H₂O)₂]·2 H₂O, [Co(L)₂(CH₃OH)₂] and (with limitations) in [Pb(L)₂·DMF]. In the remaining compounds the ligand behaves essentially as a thiolate ligand. The platinum, gold and mercury complexes [TMA]₂[Pt(L)₄], [TMA] [Au(L)₂] and [Hg(L)₂] are monomeric. In [TMA][Ag₂(L)₃]·5.5 H₂O a chain‐like structure was found. In the asymmetric unit of this structure eight silver ions, with mutual distances in the range 2.8949(4) to 3.1660(3)Å, are coordinated by twelve thiosquarato ligands. [Pb(L)₂·DMF] has also a polymeric structure. It contains a core of edge‐bridged, irregular PbS₄ polyhedra. TMA[Au(H₂NC₄O₂S)₂] has also been prepared and its structure elucidated.