The rapid component of electron paramagnetic resonance signal II: a candidate for the physiological donor to photosystem II in spinach chloroplasts.

Rapid light-induced transients in EPR Signal IIf (F-+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q minus is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E'Os.o equals to + 480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential. A model is proposed in which Q minus, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F-+). At high potentials D is oxidized in the dark, and the (Q-+F-+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

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.     

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.     

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+.     

Modulation of herbicide-binding by the redox state of Q400, an endogenous component of Photosystem II

Preincubation of isolated chloroplasts with ferricyanide, prior to addition of DCMU, unmasks a high-potential electron acceptor (Q₄₀₀) in Photosystem II that acts as an additional quencher and prolongs the fluorescence induction curve in the presence of DCMU (Ikegami, I. and Katoh, S. (1973) Plant Cell Physiol. 14, 829–836). This study confirms that Q₄₀₀ is endogenous to Photosystem II and is not a bound ferricyanide, and several new characteristics of this high potential acceptor are established. (a) It is accessible to ferricyanide even in the presence of DCMU. The rate of oxidation, however, is very slow, consistent with access only via Q_A. Accessibility may be enhanced by magnesium, reminiscent of the oxidation of Q⁻_A by ferricyanide. (b) Oxidation of Q₄₀₀ drastically suppresses the binding of DCMU at neutral and alkaline pH. Below pH 6, however, DCMU binding is essentially normal. The pH dependence of DCMU binding is consistent with the known pH dependence of the redox midpoint potential of Q₄₀₀. (c) Binding of many other inhibitors of Q_A-to-Q_B electron transfer is much less affected or even completely unaffected. These results have implications for current notions of herbicide binding and may also bear on the origin of slow phases of fluorescence induction in the presence of DCMU.     

pH dependent changes in the reactivity of the primary electron acceptor of System II in spinach chloroplasts to external oxidant and reductant

Changes in the rates of dark oxidation and reduction of the primary electron acceptor of System II by added oxidant and reductant were investigated by measuring the induction of chlorophyll fluorescence under moderate actinic light in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-inhibited chloroplasts at pH values between 3.6 and 9.5. It was found that:

1. (1) The rate of dark oxidation of photoreduced primary acceptor was very slow at all the pH values tested without added electron acceptor.

2. (2) The rate was accelerated by the addition of ferricyanide in the whole pH range. It was dependent approximately on the 0.8th power of the ferricyanide concentration.

3. (3) The rate constant for the oxidation of the primary acceptor by ferricyanide was pH-dependent and became high at low pH. The value at pH 3.6 was more than 100 times that at pH 7.8.

4. (4) The pH-dependent change in the rate constant was almost reversible when the chloroplasts were suspended at the original pH after a large pH change (acid treatment).

5. (5) An addition of carbonylcyanide m-chlorophenylhydrazone or heavy metal chelators had little effect on the rate of dark oxidation of the primary acceptor by ferricyanide.

6. (6) The dark reduction of the primary acceptor by sodium dithionite also became faster at low pH.

From these results it is concluded that at low pH the primary acceptor of System II becomes accessible to the added hydrophilic reagents even in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.     

Membrane surface potential and the reactivity of the System II primary electron acceptor to charged electron carriers in the medium

A hypothesis is proposed to explain the change in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase.Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149–166).Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

Chlorophyll radical cation in photosystem II of chloroplasts. Millisecond decay at low temperature.

We compare the absorption changes, in the near infrared and in the green part of the spectrum, induced in spinach chloroplasts suspensions, at -- 170 degrees C, by continuous light and by flashes. (1) Following flash excitation, an absorption increase peaking at 825 nm which reverses rapidly (t 1/2 = 3.0 ms) is not affected by ferricyanide; it is suppressed when chloroplasts are preilluminated in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) and hydroxylamine. The reversion of that signal is simultaneous with a partial back reoxidation of C-550 (fully reduced by the flash) and with partial (about 25%) oxidation of cytochrome b559. The magnitude of the signal peaking at 825 nm (that we attribute to the radical cation of the trap chlorophyll of Photosystem II, acting as a primary electron donor) decreases progressively within a series of successive flashes. (2) An absorption increase (40% of which is slowly reversible) with a broad peak around 810 nm is induced by continuous light or by a flash. It is suppressed by pretreatment with ferricyanide, but it is little affected by the treatment with 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea and hydroxylamine. We attribute it to oxidized P700. (3) With chloroplasts pretreated with 10 mM ferricyanide, an absorption increase, whose magnitude is nearly independent of wavelength between 790 and 870 nm, can be induced by continuous light. One saturating flash produces only 20% of the signal. This absorption change (20% of which is reversible in 30 s) might be due to a secondary donor of Photosystem II.     

Interaction of linolenic acid with bound quinone molecules in Photosystem II. Time-resolved optical and electron spin resonance studies

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.     

Oxaloacetate inhibition of succinate oxidation in tightly coupled liver mitochondria with ferricyanide as an electron acceptor

When ferricyanide is used as an artificial electron acceptor, succinate oxidation by tightly coupled liver mitochondria becomes inhibited after 1–3 min. No inhibition occurs in the presence of rotenone or glutamate establishing that oxaloacetate causes the inhibtion. Oxygen consumption by mitochondria oxidizing succinate does not become inhibited in the absence of rotenone suggesting that oxaloacetate accumulates to a greater extent when ferricyanide is added than when oxygen is the terminal acceptor. Higher levels of oxaloacetate in the ferricyanide reaction are apparently due to an increased rate of synthesis rather than a decreased rate of removal. Thus it appears that when succinate is the substrate and oxygen the terminal acceptor a control mechanism exists which blocks oxidation of malate. When ferricyanide is added as an artificial electron acceptor this control is lost and oxaloacetate accumulates to inhibit succinate oxidation.     

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.     

Studies on chlorophyll fluorescence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

1. The effect of preincubating spinach chloroplasts with ferricyanideon the time courses of chlorophyll- fluorescence in the presenceof 3-(3,4-dichlorophyl)-1,1-dimethylurea (DCMU) was studied.When DCMU was absent from the preincubation mixture, but wasadded just before the onset of excitation light, preincubationof chloroplasts with ferricyanide markedly affected the fluorescencekinetics. The rise-rate was lowered and consequently the areaabove the induction curve (S/Fv), which is proportional to thepool size of the electron acceptor(s) for photosystem 2, increased.The maximum increase in the S/Fv was attained after 3 min and10 min, respectively, of preincubation with 5?10^–4M and3?10^–5M ferricyanide.
2. When DCMU was present during preincubationwith ferricyanide,the effect of ferricyanide in increasingthe S/Fv, was completelyeliminated.
3. The effect of ferricyanidewas also suppressed by addition offerrocyanide to the preincubationmixture. The redox potentialof the ferri-ferrocyanide mixturewhich produced 50% suppressionof the ferricyanide effect wasabout 360 mV.
4. A similar dependency of the ferricyanide effecton the redoxpotential was observed in Tris-treated chloroplasts.However,the redox potential of cytochrome b-559 was markedlyloweredby Tris-treatment.
5. These results were explained byassuming the occurrence of asecondary electron acceptor, R,between the reaction centerof photosystem 2 and the DCMU-sensitivesite.

(Received February 27, 1973; )     

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron transfer between the two photosystems. II. Equilibrium constants

(1) The equilibrium constants for the redox reactions occurring between Photosystem (PS) I donors were measured on chloroplasts, dark-adapted in the presence of sodium ascorbate and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and then illuminated by d.c. light. The equilibrium constant for the electron transfer between plastocyanin and P-700 is close to 1 and the overall equilibrium constant between cytochrome f and P-700 is about 2.3. As these equilibrium constants do not depend upon the intensity of the d.c. beam, the low values we measured cannot be due to kinetic limitations. (2) The equilibrium constants were measured also in the absence of DCMU using chloroplasts in oxidizing conditions (ferricyanide or far red illumination) illuminated by a saturating flash. During the course of the reduction of PS I donors by plastoquinol molecules formed by the flash, the equilibrium constants are higher than in the preceding conditions: the value for plastocyanin to P-700 is close to 5, and that for cytochrome f to P-700 is about 25. (3) The variations of these equilibrium constants are tentatively interpreted as being due to mutual electrostatic interactions between cytochrome b and f which are included in the same complex. This model implies that the perturbation of the redox properties of cytochrome f by a positive charge located on cytochrome b is identical to the perturbation of the redox properties of cytochrome b by a positive charge located on cytochrome f.