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.     

Midpoint potential of signal II (slow) in Tris-washed photosystem-II particles

In Tris-washed Photosystem-II particles we are able to induce an EPR signal in the dark by addition of an iridium salt (K₂IrCl₆). This signal is attributed to signal II_s (slow) (D⁺) and the redox titration gives an E_m value of 760 mV for the couple $\text{D}{}^{\mathrm{+}}\text{D}$. On the basis of our previous studies on the equilibrium between D⁺Z and DZ⁺ (K = 10⁴) (Boussac, A. and Etienne, A.L. (1982) Biochem. Biophys. Res. Commun. 109, 1200–1205), we therefore attribute a value of 1 V for the E_m of the $\text{Z}{}^{\mathrm{+}}\text{Z}$ couple. A second effect of K₂IrCl₆ is to modify the spectral characteristics of signal II. We conclude that K₂IrCl₆ is able to change the environment of the species from which signal II_s and signal II_f originate.     

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 $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

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.     

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.     

Light-induced absorption changes in intact cells of Rhodopseudomonas Sphaeroides. Evidence for interaction between photosynthetic and respiratory electron transfer chains

Absorption changes, following a series of actinic flashes, linked to oxidoreduction states of ubiquinone, cytochrome c_t together with the carotenoid bandshift, have been measured for intact cells of Rhodopseudomonas sphaeroides under aerobic conditions. Binary oscillations are observed for these different contributions: (1) about one molecule of ubisemiquinone and fully reduced quinone are formed on odd and even flashes, respectively; (2) cytochrome c_t re-reduction is faster ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 50}\text{ms}$) after an even number of flashes than after an odd number; ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 100}\text{ms}$); (3) a slow-rising phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 5}\text{ms}$, antimycin A-insensitive) of the carotenoid bandshift is observed after each even flash. These results are compared to the respiratory activity of the cells under flash excitation and discussed in relation to a model, in which respiratory and photosynthetic electron chains interact at the level of cytochrome c₂ and where the terminal oxidase is supposed to have electrogenic properties.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides

The rates of electron exchange between ferricytochrome c (C^III)3 and ferrocytochrome c (C^II) were observed as a function of the concentrations of ferrihexacyanide (Fe^III) and ferrohexacyanide (Fe^II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe^II to C^III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C^III·Fe^II, in the over-all reaction:

$\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{C}{}^{\mathrm{III}}\text{·}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{C}{}^{\mathrm{II}}\text{·}\text{Fe}{}^{\mathrm{III}}\text{?}\text{k}{}_{\mathrm{?3}}\text{k}{}_3\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}$

Values for $\text{k}{}_1\text{k}{}_{\mathrm{?1}}\text{= 0.4 × 10}{}^3\text{m}{}^{\mathrm{?1}}\text{and}\text{k}{}_2\text{= 208}\text{s}{}^{\mathrm{?1}}$, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C^III in the presence of Fe^II and either KCl or Fe^III suggest that complexation is principally ionic, that Fe^III and Fe^II compete for a common site. Addition of saturating concentrations of Fe^III to C^III produced only minor changes in the nuclear magnetic resonance spectrum of C^III suggesting that complexation occurs on the protein surface.Addition of Fe^III to C^II in the presence of excess Fe^II (to retain most of the protein as C^II) increased the line width of the methyl protons of ligated methionine 80. A value for k_?2 ≈ 2.08 × 10⁴ s^?1 was calculated from the dependence of linewidth on the concentration of Fe^II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k₂ and k_?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k₂ and k_?2, respectively.     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

The role of cytochrome b-566 in the electron-transfer chain of Rhodopseudomonas sphaeroides

Spectrophotometric, kinetic, thermodynamic and stoichiometric properties of the low-potential b-type cytochrome of chromatophores from Rhodopseudomonas sphaeroides are reported. Cytochrome b-566 has a double α-band with maxima at 559 and 566 nm. Resolution of the spectrum by full-spectral redox potentiometry showed no indication that the two peaks represent more than one component. The component titrated with E_m,7 ≈ ?80 ± 10 mV. By appropriate choice of wavelength pairs and by subtraction of the contribution due to other components, the kinetics of cytochrome b-566 absorbance changes following flash excitation have been resolved from those of other components. Time-resolved flash spectra corrected for the contributions of other components are consistent with the behavior of both peaks of the α-band as a single kinetic species. The kinetics of cytochrome b-566 in the presence of antimycin show that the reduction of this cytochrome occurred only if cytochrome b-561 was reduced before the flash, either chemically, by poising the ambient redox potential (E_h) below the E_m of cytochrome b-561 (E_m,7 ≈ 50 mV), or photochemically at higher redox potentials by a previous flash. The rate of reduction of cytochrome b-566 varied with E_h. At low E_h (approx. 0 mV) reduction on the first flash showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1.25}\text{ms}$; at high E_h (approx. 180 mV) reduction on the second flash showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 10}\text{ms}$. In the absence of antimycin at E_h ≈ 0 mV, cytochrome b-566 was observed to become rapidly reduced ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 500 μ}\text{s}$) and then reoxidized ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 2}\text{ms}$) after a single flash. At higher redox potentials (E_h > 80 mV) no kinetic changes which could be unambiguously attributed to cytochrome b-566 were observed following a single flash. The results are interpreted in terms of a Q-cycle mechanism in which the reductant for cytochrome b-566 is the semiquinone formed on oxidation of ubiquinol from the quinone pool. The oxidation of the ubiquinol occurs by a concerted reaction in which one electron is accepted by the Rieske-type FeS center and the other by cytochrome b-566. We suggest that the kinetic characteristics may indicate a pathway for reduction of the b-type cytochromes in which cytochrome b-566 is the immediate electron acceptor and donates to cytochrome b-561 in a serial pathway. The experimental results in the presence of antimycin are compared with data from a computer simulation of the thermodynamic behavior of the chain, and the computer model is shown to provide an excellent fit.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

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.     

On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation $\text{k′}{}_2\text{=}\text{k}{}_{\mathrm{<mtext>b</mtext>}}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}\text{+}\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{[}\text{OH}{}^{\mathrm{?}}\text{]K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}$, where K_a is the dissociation constant of TrisH⁺. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k_b and k_a terms to k′₂. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k_b term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10² and possibly of the order 10⁶. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.