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.     

The study of the primary photoprocesses in Photosystem I of chloroplasts Recombination luminescence,chlorophyll triplet state and triplet-triplet annihilation

The dependence of the delayed luminescence of Photosystem I on the state of the reaction centers has been studied. Light flash induces a charge separation in the centers: $\text{P-700 · P-430}\textcolor{red}{}\text{P-700}{}^{\mathrm{+}}\text{· P-430}{}^{\mathrm{?}}$. Dark recombination of charges is accompanied by the recombination luminescence with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{ms}$.If the centers are in the P-700 · P-430^? state or if P-430 is inactivated by heat, then flashing of Photosystem I generates the triplet state chlorophyll with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 0.5}\text{ms}$. The triplet state has been measured by the delayed fluorescence of chlorophyll at 20 °C and 77 °K and by the chlorophyll phosphorescence at 77 °K. The delayed fluorescence at 20 °C arises from the thermal activation of the triplet state up to the excited singlet level of chlorophyll and at 77 °K it is due to triplet-triplet annihilation. The quantum yield of the triplet formation, estimated by a comparison of the light saturation curves of delayed fluorescence at 20 °C and of P-700 photooxidation under the same experimental (optical) conditions, is ≈ 0.9 of the P-700⁺ yield. Only one triplet of chlorophyll can be generated per P-700. Under heat inactivation of P-430 the triplet formation is not observed when P-700 is oxidized.It is assumed that the triplet-triplet annihilation at 77 °K is related with the strong interaction between the chlorophyll molecules in the pigment complex of Photosystem I. The possibility of a triplet participation in the primary processes of photosynthesis is discussed.     

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.     

Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein

Absorption changes accompanying the formation of light-induced P-700⁺ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700⁺ and A^?₂. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A₂ were completely lost. These results provide evidence that the intermediate electron acceptor, A₂, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A₂ is different from that of P-430.     

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.     

Photosystem II reactions in oxidant-treated chloroplast fragments.

Treatment of Photosystem II fragments with the oxidant K₂IrCl₆ destroys approximately 50% of the bulk chlorophyll and results in fragments that are twofold enriched in P680 (the Photosystem II reaction-center chlorophyll) and cytochrome b₅₅₉. The fragments retain a fully competent reaction center, as evidenced by P680 photooxidation and subsequent reduction in a back reaction with the primary electron acceptor ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5}\text{ms}$ at 25 dgK). The K₂IrCl₆-treated fragments contain no photoactive or chemically detectable C-550 and do not exhibit any variable fluorescence. These results imply that the Photosystem II primary electron acceptor is unaffected by oxidant treatment. It therefore may be concluded that neither C-550 nor the fluorescence quencher, Q, functions as the primary electron acceptor of Photosystem II.     

Analysis of absorption changes in the ultraviolet related to charge-accumulating electron carriers in Photosystem II of chloroplasts

Spinach chloroplasts were dark adapted and then submitted to a sequence of short saturating flashes. The resulting absorption changes in the near ultraviolet were analyzed and attributed to the donor and acceptor sides of Photosystem II. Our results provide a spectroscopic support to some current models of these parts of the photosynthetic electron transport.In Tris-treated chloroplasts (supplied with artificial donors) the absorption changes are largely due to the acceptor side. After each flash the signal decays with a fast phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{ms at}\text{9 °}\text{C}$) leaving a stationary level (on a 100-ms time scale). The fast phase has a small amplitude after odd-numbered flashes, whereas the stationary level behaves in a complementary fashion. The non-decaying signal is attributed mostly to the reduced secondary acceptor (A₂^?) and the fast phase to the simultaneous reoxidation of A₂^? and of the reduced primary acceptor (A₁^?). The effect of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and of redox mediators (ascorbate, ferricyanide) also support this assignment. A fraction of A₂ is shown to be reduced in dark-adapted chloroplasts, as proposed by Velthuys and Amesz (Biochim. Biophys. Acta (1974) 333, 85–94). The difference spectra support the view that A₁^? and A₂^? are plastoquinone radical anions. There are also some absorption changes that we cannot identify.In untreated chloroplasts a non-decaying absorption change (“slow phase”) occurs with a 4-flash periodicity. It is attributed to the transitions among the S states associated with the O₂-evolving complex. A fast phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{ms}$) in the decay following the first two flashes behaves like in Tris-treated chloroplasts, so that the assignment is tentatively the same. After the third flash, however, the magnitude of this fast phase is too large according to the hypothesis, so that there may be some contribution from the donor side. The fast phases become slower at lower pH (5.5 instead of 7.6), although there is no evidence for a protonation A₁^? or A₂^?.     

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.     

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.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

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.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

The role of the membrane-bound iron-sulphur centres A and B in the Photosystem I reaction centre of spinach chloroplasts

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I ($\text{P-700}$) and X.Oxidation-reduction potential titrations confirmed that Centre A had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?550}\text{mV}$, Centre B had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}\text{mV}$. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, $\text{P-700}$ photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible $\text{P-700}$ photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}$ mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.     

Quantitative relationship between photosystem I electron transport and ATP formation

The Photosystem I-dependent transport of electrons from diaminodurene to methylviologen is linear with reaction time and supports a constant rate of phosphorylation. However, if the diaminodurene is not kept fully reduced by the presence of excess ascorbate, the oxidized diaminodurene accumulates and begins to compete with the methylviologen as the electron acceptor. Thus, although the rate of ATP formation remains unchanged, an increasing proportion of the electron transport becomes cyclic and hence unmeasured. This leads to a rapid increase in the apparent efficiency of phosphorylation which is misleading.In contrast, it is known that the oxidized form of 3,3′-diaminobenzidine polymerizes to form an insoluble substance which should not be available to serve as an electron acceptor. However, 3,3′-diaminobenzidine is not a satisfactory donor of electrons in Photosystem I reactions for two reasons: the rate of electron transport quickly falls with reaction time and the oxidized form of 3,3′-diaminobenzidine seems to be an exceptionally efficient electron acceptor near the beginning of the period of illumination when it is presumably not yet polymerized. Thus in the first 2–3 sec of illumination when the reaction is still rapid much of the electron transport is cyclic and therefore unmeasured, especially in the absence of excess ascorbate. This cycling of electrons, which leads to an inflated apparent efficiency ($\text{P}\text{e}{}_2\text{> 2}$), is particularly pronounced at low donor concentrations.When cyclic electron transport is avoided by the use of ascorbate or by the selection of appropriate reaction times, both diaminodurene and 3,3′-diaminobenzidine support phosphorylation with an efficiency which is approximately half of the efficiency exhibited by the overall Hill reaction. The same is true when 2,5-diaminotoluene, tetrachlorohydroquinone, 4,5-dimethyl-o-phenylenediamine, and reduced 2,6-dichloroindophenol serve as electron donors. With these six substances, the phosphorylation efficiences were 0.57 ± 0.1 molecules of ATP formed for each pair of electrons transferred ($\text{P}\text{e}{}_2$). In the same chloroplasts preparations, the transport of electrons from water to methylviologen-supported phosphorylation with a $\text{P}\text{e}{}_2$ of 1.2.     

The composition-dependence of the extent of reaction between a multivalent acceptor and a bivalent ligand: Implications of self-interaction of the ligand in precipitin effects

Theory is presented relating to the reversible interaction of an f-valent acceptor, A, with a bivalent ligand, B, which leads to the formation of a series of complexes comprising networks of alternating A and B molecules. An explicit expression is derived for the overall extent of reaction in terms of the total molar concentrations of reactants $\text{(}\text{m}{}_{\mathrm{A}}$ and $\text{m}{}_{\mathrm{B}}\text{)}$, the valency of the acceptor and the site-binding constant, k, governing the equilibria. It is shown by differentiation of this expression holding $\text{m}{}_{\mathrm{A}}$ (or $\text{m}{}_{\mathrm{B}}$) fixed that relations are available for the independent evaluation of f and k from a combination of precipitin and radioimmunoassay experiments. Moreover, it is established that dilution with solvent ($\text{m}{}_{\mathrm{A}}\text{/}\text{m}{}_{\mathrm{B}}$ fixed) cannot lead to the appearance of a precipitate with this type of crosslinking system. The latter observation forms the background for the development of theory pertaining to the joint operation of ligand dimerization, 2B?B₂, and crosslinking of the multivalent acceptor with bivalent B₂. The theoretical examination of this system is developed in terms of site-probability functions and involves the delineation of unique solutions for the extent of crosslinking reaction aided by the definition of the extent of binding in defined limits. It is shown with the use of numerical examples that the system involving self-associating ligand may result in the appearance of a precipitate on dilution with solvent and the conditions for the operation of this phenomenon are elucidated. It is noted that other types of ligand self-interaction may lead to similar effects in crosslinking systems, and the general principles emerging from this study are discussed in terms of systems in which antibody ligands are known to be involved in association reactions or are suspected to be so involved on the basis of precipitation effects observed on dilution with solvent.