Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I-, Fe(CN)6(4-), W(CN)8(4-) are poor reducing agents for Z.+ whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors. A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z.+. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner surface.     

Surface charge asymmetry and a specific calcium ion effect in chloroplast photosystem II

We have used the decay kinetics of Signal IIf in Tris-washed chloroplasts as a direct probe to reactions on the oxidizing side of Photosystem II. A study of the salt concentration dependence of the rate of reduction of Z . + by the ascorbate monoanion has been interpreted by using the Gouy-Chapman diffuse double layer model and allows the calculation of an inner membrane surface charge density of -3.4 +/- 0.3 microC . cm-2 at pH = 8.0 in the vicinity of Photosystem II. We have also measured the outer membrane surface charge density at this pH in Tris- and sucrose-washed chloroplasts by monitoring the rate of potassium ferricyanide oxidation of Q-, and arrive at values of -2.2 +/- 0.3 microC . cm-2 and -2.1 microC . cm-2, respectively. From these experiments we conclude that in dark-adapted chloroplasts at pH 8.0 there exists a transmembrane electric field in the vicinity of Photosystem II which arises from this surface charge asymmetry. In the presence of 10 mM monovalent salts, the transmembrane potential difference is of the order of 20 mV, corresponding to a field of 4 . 10(4) V . cm-1 (negative inside) for a 50A membrane. It is both smaller in magnitude and in the opposite direction compared to the photoinduced transmembrane field which gives rise to the 515 nm absorption change. We have also found non-double layer Ca2+ effects on the decay kinetics of Signal IIf with both charged (ascorbate monoanion) and neutral (diphenylcarbazide) donors. These results suggest a change in the environment of Z from lipophilic to hydrophilic upon specific binding of Ca2+.     

NMR relaxation studies of intracellular Na in red blood cells

The state of intracellular Na⁺ in human and dog erythrocytes was characterized by ²³Na-NMR using dysprosium complexes as shift reagents. Intracellular Na⁺ concentrations were determined using integration of the inner Na⁺ NMR signals and measurements of the intracellular volume using ⁵⁹Co-NMR of extracellular Co(CN)³⁻₆. T₂ was found to be significantly shorter than T₁, indicating some binding to macromolecules. While the longitudinal magnetization decay follows a single exponential the transverse magnetization could be fitted with a double-exponential function. It was shown that neither the binding to the inner side of the membrane nor binding to hemoglobin contributes to the relaxation enhancement.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Absorbance difference spectra upon charge transfer to secondary donors and acceptors in Photosystem II

Charge-transfer reactions to secondary electron donors (Z, M) and acceptors (Q_A, Q_B) in Photosystem II particles isolated from a thermophilic cyanobacterium Synechococcus sp. (Schatz, G.H. and Witt H.T. (1984) Photobiochem. Photobiophys. 7, 1–14) were analyzed by measurements of fluorescence yield and absorbance changes in the millisecond time domain induced by repetitive flashes. (1) The electron-transfer reaction Q⁻_AQ_B → Q_AQ⁻_B was found to occur with kinetic phases of 0.2 ± 0.1 ms and 1.5 ± 0.5 ms half-time. At 10 ms after flashes an equilibrium distribution of Q⁻_AQ_B/Q_AQ⁻_B of about 15/85 in oxygen-evolving and of about 25/75 in Tris-treated PS II particles was reached. (2) The absorbance difference spectra were determined for (Q⁻_A - Q_A), (Q⁻_B - Q_B), (Z⁺ - Z) and for (S₄ - S₀), the transition associated with oxygen evolution. In the ultraviolet region they show that these electron-acceptors and -donors are the same as in spinach PS II. In the visible region all the difference spectra contain major contributions by electrochromic bandshifts due to electrostatic interaction of the reduced acceptors or oxidized donors with nearby reaction center pigments. Upon electron transfer from Q⁻_A to Q_B electrochromic bandshifts due to interaction with pheophytin a disappeared almost completely. Bandshifts observed in the (Z⁺ - Z) and (S₄ - S₀) spectra were attributed to chlorophyll a.     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

Two electron donation sites for exogenous reductants in chloroplast photosystem II.

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20-40%) in Signal IIf magnitude accompanying donor addition at low (smaller than 10(-%) M) concentrations, but the extent of the decrease does not change further with increasing donor concentrations. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration. In the presence of the hydrophilic donor, Mn-2+, the Signal IIf decay halftime remains constant with increasing Mn-2+ concentration. However, the flash-induced Signal IIf magnitude pregressively decreases with increasing Mn-2+ concentration. These results are interpreted in terms of two competing paths for the reduction of P680+. In one path P680+ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn-2+ reduces P680+ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

Identification of the 120 μs phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH

After a 500 μs laser flash a 120 μs phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Biophys. Acta 376, 329–344, indicates that their EPR signal II_f which they suppose to be due to Z⁺, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 μs phase. We explain our 120 μs phase by the intrinsic back reaction of the excited reaction center in the presence of Z⁺, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333–342. The redox state of Z⁺ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the μs region are compared with those obtained in the ms region.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH₃CH(OH), CO₂, O₂, and e⁻_aq′ and the oxidation of the reduced cytochrome c derivatives by Fe(CN)³⁻₆ were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partitioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

Two electron donation sites for exogenous reductants in chloroplast Photosystem II

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20–40%) in Signal IIf magnitude accompanying donor addition at low (< 10^?5M) concentrations, but the extent of the decrease does not change further with increasing donor concentration. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration.In the presence of the hydrophilic donor, Mn²⁺, the Signal IIf decay halftime remains constant with increasing Mn²⁺ concentration. However, the flash-induced Signal IIf magnitude progressively decreases with increasing Mn²⁺ concentration.These results are interpreted in terms of two competing paths for the reduction of P680⁺. In one path P680⁺ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn²⁺ reduces P680⁺ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

Studies on a thermal reaction associated with photosynthetic oxygen evolution

The modulated polarographic technique of O₂ detection was applied to Chlorella to study the rate-limiting thermal reaction between Photosystem II and O₂ evolution. From an analysis of the operation of the polarograph at different frequencies, it was concluded that a first order thermal reaction of rate constant 305±20 (S.E.) s⁻¹ was consistent with the results of 22 °C. When the algae were successively studied in solutions made up with ²H₂O and H₂O, a kinetic isotopic effect for the rate constant of 1.29±0.05 (S.E.) was found. This suggests that the rate limiting step does not involve the breaking of the O-H bond in water. A temperature study of the rate constant indicated an activation energy of 5.9±0.5 (S.E.) kcal·mole⁻¹ and an entropy of activation of −25 cal·degree⁻¹·mole⁻¹. The linearity of the Arrhenius plot between 8 and 42 °C demonstrated that only one reaction was rate-limiting over this temperature range.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^-₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^-₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^-₂. Second order kinetics for the decay of RNO^-₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^-₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

Absorption studies of Photosystem I photochemistry in the absence of vitamin K-1

Flash-induced absorption changes were studied on different timescales (nanosecond to millisecond) and at different temperatures (10 to 278 K) in highly enriched spinach PS I particles lacking vitamin K-1 and in which the electron transfer from the primary acceptor to the secondary acceptors was blocked. At all temperatures, the initial absorption change at 820 nm was followed by a fast decay (t_1/2 ≈ 47 ns at 278 K and ≈ 82 ns at 10 K) which is attributed to the decay of the primary radical pair (P-700⁺-A⁻₀). A slower phase of absorption decay is attributed to the P-700 triplet state, which was formed as a result of the biradical recombination, with a yield of about 30% at 278 K and about 75% at 10 K. Under air, the ³P-700 state decayed with a t_1/2 of about 50 μs at 278 K, whereas in the absence of oxygen it decayed with t_1/2 ≈ 560 μs. At 278 K, this yield was shown to depend on the presence of a magnetic field, with a maximum around 60 G. The ³P-700 decay halftime was nearly independent of temperature in the absence of oxygen (t_1/2 ≈ 1 ms at 10 K). The implications for the mechanisms involved in this decay are discussed. Addition of vitamin K-1 to these particles resulted in a decrease in the amplitude of the fast submicrosecond decay and a concomitant increase in the amplitude of a slow phase, indicating an efficient transfer from A⁻₀ to vitamin K-1. However, most functional properties of the acceptor A₁ were not reconstituted under these conditions.     

Kinetics of appearance and disappearance of light-induced EPR signals of P700 and iron-sulfur protein(s) at low temperature

Light-induced changes of EPR signals in Photosystem-I subchloroplast particles at temperatures between 225 and 13 °K showed that the rates of onset of photooxidation of P700 and photoreduction of iron-sulfur protein(s) are identical and instantaneous within the limits of resolution of our instruments. The fraction of the P700⁺ EPR signal that appears reversibly decreased with decreasing temperature down to 13 °K when the photoreaction was completely irreversible. At temperatures below 225 °K, the reversible fraction consists of two approximately equal portions with decay halftimes of approx. 3 and 75 s, respectively. Light-induced absorption changes due to P700 photooxidation at low temperatures monitored at 700 nm showed a similar kinetic pattern.

Since the reduced iron-sulfur protein signals can only be detected at very low temperature, their decay kinetics cannot be continuously monitored at higher temperatures. Therefore, exposure at appropriate temperatures and reaction times were selected according to the decay kinetics of P700⁺, after which decay was stopped by lowering the temperature to 13 °K and the P700⁺ and reduced iron-sulfur protein signals were recorded and compared. In the temperature range (225-13 °K) studied, the decay of P700⁺ and reduced iron-sulfur protein signals appears identical, suggesting that the two oppositely charged species recombine in the dark. These experiments support the view that iron-sulfur protein(s) is the reaction partner of P700 in the primary photochemical act of Photosystem I.     

The versatile coordination chemistry of tricyanoethenolate, [(NC)2C=C(CN)O]

The reaction of meso-tetrakis (4-dimethoxyphenyl) porphinatomanganese(II), MnTP_OMeP, with TCNE (TCNE = tetracyanoethylene) leads to the formation of [MnTP_OMeP]⁺ [TCNE]⁻ and [MnTP_OMeP]⁺[OC(CN)C(CN)₂]⁻. The single-crystal X-ray structures of the latter as well as [Cu(bipy)₂Cl]⁺ [OC(CN)C(CN)₂]⁻ were determined. The former has a disordered [OC(CN)C(CN)₂]⁻ bridging via C and O between a pair of Mn^III sites, whereas the latter has an isolated [OC(CN)C(CN)₂]⁻ unbound to Cu^II. The IR characterization for μ₂-C,O bound [OC(CN)C(CN)₂]⁻ is at 2219m and 2196s (νCN) cm⁻¹ and at 1558s (νCO) cm⁻¹ while for unbound [OC(CN)C(CN)₂]⁻ it is at 2210m, 2203m, 2181m (νCN) cm⁻¹ and at 1583s (νCO) cm⁻¹.     

Identification of the 120 mus phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH.

After a 500 mus laser flash a 120 mus phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Bio-phys. Acta 376, 329-344, indicates that their EPR signal IIf which they suppose to be due to Z+, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 mus phase. We explain our 120 mus phase by the intrinsic back reaction of the excited reaction center in the presence of Z+, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333-342. The redox state of Z+ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the mus region are compared with those obtained in the ms region.     

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e⁻_aq) and carboxyl anion radical (CO₂) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e⁻_aq with the negatively charged ferricytochrome c-551 (17 nM⁻¹ · s⁻¹) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM⁻ · s⁻). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e⁻_aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10⁴ s⁻¹) observed after fast e⁻_aq reduction is attributed to a small conformational change. The rate of reaction of CO₂ with ferricytochrome c-551 (0.7 nM⁻¹ · s⁻) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.     

A nitrate reductase activity in Rhodopseudomonas capsulata linked to electron transfer and generation of a membrane potential

We have isolated from a laboratory strain of Rhodopseudomonas capsulata a spontaneous mutant possessing a dissimilatory NO⁻₃ reductase activity. Reduction of NO⁻₃ under dark and anaerobic conditions generated a membrane potential, and was inhibited by rotenone, oxygen and illumination.