Reactions between primary and secondary acceptors of Photosystem II in Chlorella Pyrenoidosa under anaerobic conditions as studied by chlorophyll a fluorescence

The kinetics of the fluorescence yield Ф of chlorophyll a in Chlorella pyrenoidosa were studied under anaerobic conditions in the time range from 50 μs to several minutes after short ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{ns}$ or 5 μs) saturating flashes. The fluorescence yield “in the dark” increased from $\text{Ф = 1}$ at the beginning to $\text{Ф ≈ 5}$ in about 3 h when single flashes separated by dark intervals of about 3 min were given.After one saturating flash, Ф increased to a maximum value (4–5) at 50 μs, then Ф decreased to about 3 with a half time of about 10 ms and to the initial value with a half time of about 2 s. When two flashes separated by 0.2 s were given, the first phase of the decrease after the second flash occurred within 2 ms. After one flash given at high initial fluorescence yield, the 10-ms decay was followed by a 10 s increase to the initial value. After the two flashes 0.2 s apart, the rapid decay was not follewed by a slow increase.These and other experiments provided additional evidence for and extend an earlier hypothesis concerning the acceptor complex of Photosystem II (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250–256; Velthuys, B. R. and Amesz, J. (1974) Biochim. Biophys. Acta 333, 85–94): reaction center 2 contains an acceptor complex QR consisting of an electron-transferring primary acceptor molecule Q, and a secondary electron acceptor R, which can accept two electrons in succession, but transfers two electrons simultaneously to a molecule of the tertiary acceptor pool, containing plastoquinone (A). Furthermore, the kinetics indicate that 2 reactions centers of System I, excited by a short flash, cooperate directly or indirectly in oxidizing a plastohydroquinone molecule (A^2?). If initially all components between photoreaction 1 and 2 are in the reduced state the following sequence of reactions occurs after a flash has oxidised A^2? via System I: Q^?R^2? + A → Q^?R + A^2? → QR^? + A^2?. During anaerobiosis two slow reactions manifest themselves: the reduction of R (and A) within 1 s, presumably by an endogenous electron donor D₁, and the reduction of Q in about 10 s when R is in the state R^? and A in the state A^2?. An endogenous electron donor, D₂, and Q^? compete in reducing the photooxidized donor complex of System II in reactions with half times of the order of 1 s.     

Flash-induced carotenoid radical cation formation in Photosystem II

Light excitation of chloroplasts at low temperature produces absorption changes (ΔA) with a large positive peak at 990 nm and a bleaching around 480 nm. ΔA at 990 nm rises with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.6}\text{ms}$ at 20–77 K and remains largely stable. This signal is not observed when Photosystem II (PS II) photochemistry is blocked by reduction of the primary plastoquinone. It is observed also in purified PS II particles, in which case it could be shown that during a sequence of short flashes, the absorption at 990 nm rises in parallel with plastoquinone reduction measured at 320 nm. In chloroplasts the light-induced 990-nm ΔA at 77 K is increased under oxidizing conditions (addition of ferricyanide) and upon addition of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p). At 21°C, flash excitation of chloroplasts or of PS II particles induces only a very small ΔA at 990 nm, even when this is measured with a 100-ns time resolution or when the material is preilluminated. In both materials, however, a large flash-induced ΔA takes place when various lipophilic anions are added. After a flash the signal rises in less than 100 μs and its decay varies with experimental conditions; the decay is strongly accelerated by benzidine. The difference spectrum measured in PS II particles includes a broad peak around 990 nm and a bleaching around 490 nm. These absorption changes are attributed to a carotenoid radical cation formed at the PS II reaction center. It is estimated that in the presence of lipophilic anions at room temperature, one cation can be formed by a single flash in 80% of the reaction centers. At cryogenic temperature approx. 8% of the PS II reaction centers can oxidize a carotenoid after a single flash.     

A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 μs to 20 μs after a short saturating flash ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25}\text{ns}$) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 μs luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ^? before the flash, about one third for centers in the state S⁺PQ^? or SPQ before the flash, and about one tenth for centers in the state S⁺PQ before the flash. S is the secondary donor complex with zero charge; S⁺ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q^? is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two.The 1 μs decay of the luminescence is probably caused by the disappearance of P⁺ formed in the laser flash according to the reaction ZP⁺ → Z⁺P in which Z is the molecule which donates an electron to P⁺ and which is part of S. After addition of hydroxylamine, the 1 μs luminescence component changed with the incubation time exponentially (τ = 27 s) into a 30 μs component; during the same time, the variable fluorescence yield, measured 9 μs after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 μs luminescence increased about 50% with the same time constant (τ = 7 min). Heat treatment abolished both luminescence components.The 1 μs luminescence component saturated at about the same energy as the System II fluorescence yield 60 μs after the laser flash and as the slower luminescence components. From the observation that the amplitude is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ^?), we conclude that the 1 μs luminescence is probably caused by the reaction

$\text{PWQ}{}^{\mathrm{?}}\text{+hv →}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→}\text{P}{}^{\mathrm{+}}\text{W}{}^{\mathrm{?}}\text{Q}{}^{\mathrm{?}}\text{→}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→ PWQ}{}^{\mathrm{?}}\text{+hv}$

in which W is an acceptor different from Q. The presence of S⁺ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides: A photoselection study

The relative orientation of the pigments of reaction centers from Rhodopseudomonas sphaeroides has been studied by the photoselection technique.A high value (+0.45) of $\text{p =}\text{(ΔA}{}_{\mathrm{<mtext>V</mtext>}}\text{? ΔA}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{(ΔA}{}_{\mathrm{<mtext>V</mtext>}}\text{+ ΔA}{}_{\mathrm{<mtext>H</mtext>}}$) is obtained when exciting and observing within the 870 nm band which is contradictory to the results of Mar and Gingras (Mar, T. and Gingras, G. (1976) Biochim. Biophys. Acta 440, 609–621) and Shuvalov et al. (Shuvalov, V.A., Asadov, A.A. and Krakhmaleva, I.N. (1977) FEBS Lett. 76, 240–245). It is shown that the low values of p obtained by both groups were erroneous due to excitation conditions.Analysis of the polarization of light-induced changes when exciting with polarized light in single transitions (spheroiden band and bacteriopheophytin Q_x bands) enable us to propose a possible arrangement of the pigments within the reaction center. It is concluded that the 870 nm band corresponds to a single transition and is one of the two bands of the primary electron donor (P-870). The second band of the bacteriochlorophyll dimer is centred at 805 nm. The Q_y transitions of the molecules constituting the bacteriochlorophyll dimer are nearly parallel (angle less than 25°).The two bacteriopheophytin molecules present slightly different absorption spectra in the near infra-red. Both bacteriopheophytin absorption bands are subject to a small shift under illumination. The angle between the Q_y bacteriopheophytin transitions is 55° or 125°. Both Q_y transitions are nearly perpendicular to the 870 nm absorption band. Finally, the carotenoid molecules makes an angle greater than 70° with the 870 nm band and the other bacteriochlorophyll molecules.     

Properties of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.: Linear dichroism and low temperature spectra

Methods of preparing dried gelatin films containing purified reaction centers of Rhodopseudomonas sphaeroides are described. The spectral properties of reaction centers in solution are essentially maintained in dried gelatin films. These films are uniform and have excellent optical properties, showing little particulate scattering at temperatures down to about 4K. Film contraction on cooling to 90K is less than 1% in linear dimension. Linear dichroism spectra are reported for films at room and low temperature. Reaction centers show a moderate amount of linear dichroism in unstretched gelatin films; the magnitude of the linear dichroism becomes much greater when the films are stretched. In stretched films, linear dichroic ratios ($\text{A}{}_{\mathrm{\parallel }}\text{A}{}_{\mathrm{\perp }}$; absorbance measured with electric vector parallel and perpendicular to stretching direction) between 1.7 and 2.2 were obtained for the 860 nm absorption band of the bacteriochlorophyll component that undergoes primary photooxidation. The relative polarizations of light-induced absorption changes of reaction centers in stretched films are similar to those reported by Vermeglio and Clayton ((1976) Biochim. Biophys. Acta 449, 500–515) and support their hypothesis that absorbance decreases, maximal near 860 and 810 nm, and an increase near 790 nm are associated with the respective disappearance and appearance of discrete bands characteristic of the reduced and oxidized bacteriochlorophyll dimer. This interpretation is also supported by the polarization of the absolute absorption spectrum near 810 and 860 nm. An absorption band near 540 nm, ascribed to the Q_x transitions of two molecules of bacteriopheophytin in the reaction center, is split at low temperatures into two bands having similar polarizations. This splitting is probably not due to exciton coupling of the two molecules, since excition theory predicts different polarizations.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Molar extinction coefficients and other properties of an improved reaction center preparation from Rhodopseudomonas viridis

Reaction centers have been purified from chromatophores of Rhodopseudomonas viridis by treatment with lauryl dimethyl amine oxide followed by hydroxyapatite chromatography and precipitation with ammonium sulfate. The absorption spectrum at low temperature shows bands at 531 and 543 nm, assigned to two molecules of bacteriopheophytin b. The 600 nm band of bacteriochlorophyll b is resolved at low temperature into components at 601 and 606.5 nm. At room temperature the light-induced difference spectrum shows a negative band centered at 615 nm, where the absorption spectrum shows only a weak shoulder adjacent to the 600 nm band. The fluorescence spectrum shows a band at 1000 nm and no fluorescence corresponding to the 830 nm absorption band. Two molecules of cytochrome 558 and three of cytochrome 552 accompany each reaction center. The differential extinction coefficient (reduced minus oxidized) of cytochrome 558 at 558 nm was estimated as 20 ± 2 mM^?1 · cm^?1 through a coupled reaction with equine cytochrome c. The extinction coefficient of reaction centers at 960 nm was determined to be 123 ± 25 mM^?1 · cm^?1 by measuring the light-induced bleaching of P-960 and the coupled oxidation of cytochrome 558. The corresponding extinction coefficient at 830 nm is 300 ± 65 mM^?1 · cm^?1. The absorbance ratio $\text{a}{}_{\mathrm{<mtext>280</mtext><mtext>nm</mtext>}}\text{a}{}_{\mathrm{<mtext>830</mtext><mtext>nm</mtext>}}$ in our preparations was 2.1, and there was 190 kg protein per mol of reaction centers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major components of apparent molecular weights 31 000, 37 000 and 41 000.     

Quenching de la chlorophylle in vivo par le m-dinitrobenzene

The quenching of fluorescence and inhibition of photochemical activities by m-dinitrobenzene have been studied in unicellular algae and chloroplasts.The complementary area $\text{S(=}\text{∫}\text{0}\text{∞}\text{(Δφ}{}_{\mathrm{<mtext>M</mtext>}}\text{?Δφ)}\text{d}\text{t)}$ is decreased in the same fashion as the maximum amplitude of the variable fluorescence Δφ_M, suggesting the invariance of S properly normalized by Δφ_M. A photochemical type inhibition for all photochemical activities (oxygen evolution, Photoreaction II and I) is observed in a concentration range higher than that required to quench Δφ_M. The ratio of the photochemical rate in limiting light to the O₂ burst elicited by a flash is constant whatever the level of inhibition. The pattern of oscillation of O₂ burst during a sequence of flashes is also unmodified, the amplitude only being decreased.In order to explain these results, it is assumed that dinitrobenzene (DNB) has a quenching effect both on the center-chlorophyll and the collector-chlorophyll of the System II photosynthetic units; when the external quencher is only acting on the collector, the trapping efficiency for the center is unmodified, but, when the center is turned into its inactive form by the photochemical reaction, the fluorescence of the collector is quenched. It is shown that the rule of invariance of the normalized complementary area applies to this type of quenching; accordingly, the zero level of the System II fluorescence, within the constant part, (cf. Lavorel, J. et Joliot, P. (1972) Biophys. J. 12, 815–831) should lie close to the 0 level (dark-adapted state).     

Development of the photosystem II unit in plastids of bean leaves greened in periodic light

The photosystem II (PS II) unit formation and development, as monitored by the kinetics of the fluorescence induction, was studied in greening protochloroplasts isolated from etiolated bean leaves exposed to periodic light-dark cycles (LDC). It was found that: (i) The protochloroplasts show the well-known biphasic induction. The $\text{F}{}_{\mathrm{<mtext>MAX</mtext>}}\text{F}{}_{\mathrm{<mtext>O</mtext>}}$ ratio increases with increasing exposure to LDC, and values almost twice as high as those of mature chloroplasts are reached. The fluorescence yield increases still more by the addition of NH₂OH. (ii) The ratio ($\text{F}{}_{\mathrm{<mtext>MAX</mtext>}}\text{-F}{}_{\mathrm{<mtext>O</mtext>}}\text{)}\text{F}{}_{\mathrm{<mtext>MAX</mtext>}}$, representing the yield of primary photochemistry, reaches values much higher than those of mature chloroplasts. (iii) The rate of fluorescence rise, in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-di-methylurea (DCMU), is at least seven times slower than that of mature chloroplasts, and it remains constant during exposure to LDC. (iv) The shape of the fluorescence kinetics is exponential early during exposure to LDC but later it becomes sigmoidal, indicating the development of energy transfer between PS II units, (v) Dark incubation after a number of LDC increases the $\text{F}{}_{\mathrm{<mtext>MAX</mtext>}}\text{F}{}_{\mathrm{<mtext>O</mtext>}}$ ratio without changing the rate of the fluorescence rise, (vi) Transfer of the plants from LDC to continuous illumination induces a decrease in the $\text{F}{}_{\mathrm{<mtext>MAX</mtext>}}\text{F}{}_{\mathrm{<mtext>O</mtext>}}$ ratio and an increase in the rate of the fluorescence rise. The results indicate that initially small PS II units are formed, which contain mainly the reaction center with a few chlorophyll a molecules closely packed around it. At the same time H₂O-splitting enzymes are synthesized which, however, are light activated. These small units are very efficient for photochemistry. As the number of small units increases, aggregates are formed, which seem to have the reaction centers very close to each other. The aggregation of the units is controlled by the structural development and organization of the membrane and not by the concentration and type of chlorophyll. The excess chlorophyll formed after further exposure to continuous illumination is inserted into preexisting units, thus increasing their size and making them more efficient in absorbing the incident light.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Two photocycles in halobacterium halobium that lacks bacteriorhodopsin

The photochemical reactions in bacteriorhodopsin-free mutant (bR^?) of Halobacterium halobium (JW-1) membranes have been studied using flash photolysis. Two photocycles were found in envelope vesicles as well as in a membrane fragment from (JW-1). A pigment absorbing at ca. 590 nm undergoes a faster photocycle, with a phototransient at ca. 500 nm ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10 ms}$). A second pigment absorbing at ca. 580 nm undergoes a slower photocycle, accompanying a phototransient absorbing below 410 nm ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 0.8 s}$).     

The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y₄) is lower compared to Y₃ than at lower flash frequency. At 4 Hz, the calculated S₀ concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent $\text{S}{}_0\text{(}\text{S}{}_0\text{+}\text{S}{}_1\text{)}$ ratio increases to about 0.2. This is explained by a relatively fast donation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.0–1.5}\text{s}$) of one electron by an electron donor to S₂ and S₃ in 15–25% of the Photosystem II reaction chains. The one-electron donor to S₂ and S₃ appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal II_s. (ii) The probability for the fast one-electron donation to S₂ and S₃ has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S₂ decay becomes 10-fold faster ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5–6}\text{s}$) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S₂ decay was extremely slow. The S₃ decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S₂ is reduced mainly by Q^?_A, whereas S₃ is not. (iii) In the absence of CO₂/HCO^?_A and in the presence of formate, the fast one-electron donation to S₂ and S₃ does not occur. Addition of HCO^?₃ restores the fast decay of part of S₂ and S₃ to almost the same extent as in control thylakoids. The slow phase of S₂ and S₃ decay is not influenced significantly by CO₂/HCO^?₃. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q^?_A oxidation without interference of Q_B, were 2.3-fold slower in the absence of CO₂/HCO^?₃ than in its presence. (iv) An almost 3-fold decrease in decay rate of S₂ is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q^?_A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q^?_B is responsible for this shift of equilibrium Q^?_A concentration, these data would suggest that the pK_a value for Q^?_B protonation is somewhat higher than 7.6, assuming that the protonated form of Q^?_B cannot reduce Q_A.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

The existence of a high photochemical turnover rate at the reaction centers of System II in Tris-washed chloroplasts

In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of $\text{≈ 1 μs}$. It has been found that X 320 is reduced by a flash in $\text{? 1 μs}$. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.     

The type,amount, location,and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7′,8′-tetrahydro-ψ,ψ-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1,2,7′,8′-tetrahydro-ψ,ψ-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm ($\text{Q}{}_{\mathrm{<mtext>x</mtext>}}$) transition of the bacteriochlorophyll complex.