The orientation of membrane bound radicals: an EPR investigation of magnetically ordered spinach chloroplasts.

The orientation of membrane-bound radicals in spinach chloroplasts is examined by electron paramagnetic resonance (EPR) spectroscopy of chloroplasts oriented by magnetic fields. Several of the membrane-bound radicals which possess g-tensor anisotropy display EPR signals with a marked dependence on the orientation of the membranes relative to the applied EPR field. The fraction of oxidized and reduced plastocyanin, P-700, iron-sulfur proteins A and B, and the X center, an early acceptor of Photosystem I, can be controlled by the light intensity during steady-state illumination and can be trapped by cooling. The X center can be photoreduced and trapped in the absence of strong reductants and high pH, conditions previously found necessary for its detection. These results confirm its role as an early electron acceptor in P-700 photo-oxidation. X is oriented with its smallest principal g-tensor axis (gx) predominantly parallel to the normal to the thylakoid membrane, the same orientation as was found for an early electron acceptor based on time-resolved electron spin polarization studies. We propose that the X center is the first example of a high potential iron-sulfur protein which functions in electron transfer in its 'superreduced' state. We present evidence which suggests that iron-sulfur proteins A and B are 4Fe-4S clusters in an 8Fe-8S protein. Center B is oriented with gy predominantly normal to the membrane plane. The spectra of center A and plastocyanin do not show significant changes with sample orientation. In the case of plastocyanin, this may indicate a lack of molecular orientation. The absence of an orientation effect for reduced center A is reconcilable with a 4Fe-4S geometry, provided that the electron obtained upon reduction can be shared between any pair of Fe atoms in the center. Orientation of the 'Rieske' iron-sulfur protein is also observed. It has axial symmetry with g parallel close to the plane of the membrane. A model is proposed for the organization of these proteins in the thylakoid membrane. A new EPR signal was observed in oriented chloroplasts. This broad unresolved resonance displays a g value of 3.2 when the membrane normal is parallel to the field. It shifts to g = 1.9 when the membrane normal is perpendicular to the field. The signal is sensitive to illumination and to washing of the thylakoid membranes of broken chloroplasts. We suggest that there is a relation between this signal and the water-oxidizing enzyme system.     

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Electron spin polarization in photosynthesis and the mechanism of electron transfer in photosystem I. Experimental observations.

Transient electron paramagnetic resonance (EPR) methods are used to examine the spin populations of the light-induced radicals produced in spinach chloroplasts, photosystem I particles, and Chlorella pyrenoidosa. We observe both emission and enhanced absorption within the hyperfine structure of the EPR spectrum of P700+, the photooxidized reaction-center chlorophyll radical (Signal I). By using flow gradients or magnetic fields to orient the chloroplasts in the Zeeman field, we are able to influence both the magnitude and sign of the spin polarization. Identification of the polarized radical and P700+ is consistent with the effects of inhibitors, excitation light intensity and wavelength, redox potential, and fractionation of the membranes. The EPR signal of the polarized P700+ radical displays a 30% narrower line width than P700+ after spin relaxation. This suggests a magnetic interaction between P700+ and its reduced (paramagnetic) acceptor, which leads to a collapse of the P700+ hyperfine structure. Narrowing of the spectrum is evident only in the spectrum of polarized P700+, because prompt electron transfer rapidly separates the radical pair. Evidence of cross-relaxation between the adjacent radicals suggests the existence of an exchange interaction. The results indicate that polarization is produced by a radical pair mechanism between P700+ and the reduced primary acceptor of photosystem I. The orientation dependence of the spin polarization of P700+ is due to the g-tensor anisotropy of the acceptor radical to which it is exchange-coupled. The EPR spectrum of P700+ is virtually isotropic once the adjacent acceptor radical has passed the photoionized electron to a later, more remote acceptor molecule. This interpretation implies that the acceptor radical has g-tensor anisotropy significantly greater than the width of the hyperfine field on P700+ and that the acceptor is oriented with its smallest g-tensor axis along the normal to the thylakoid membranes. Both the ferredoxin-like iron-sulfur centers and the X- species observed directly by EPR at low temperatures have g-tensor anisotropy large enough to produce the observed spin polarization; however, studies on oriented chloroplasts show that the bound ferredoxin centers do not have this orientation of their g tensors. In contrast, X- is aligned with its smallest g-tensor axis predominantly normal to the plane of the thylakoid membranes. This is the same orientation predicted for the acceptor radical based on analysis of the spin polarization of P700+, and indicates that the species responsible for the anisotropy of the polarized P700+ spectrum is probably X-. The dark EPR Signal II is shown to possess anisotropic hyperfine structure (and possibly g-tensor anisotropy), which serves as a good indicator of the extent of membrane alignment.     

The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

Evidence for only one iron-sulfur cluster in center X of photosystem I from higher plants

In the photosystem I of thylakoid membranes, the photoinduced electron transfer involves three iron-sulfur centers, A, B, and X. Among them, center X is characterized by very unusual spectroscopic and redox properties. Recent arguments have been presented in favor of a [2Fe-2S] structure for the clusters implicated in this center, but the number of these clusters is still a controversial question. By using an original EPR method, based on the differences in the relaxation properties of A, B, and X, we have determined the stoichiometry for the iron-sulfur clusters in photosystem I. Our measurements indicate that center X is composed of a single iron-sulfur cluster per P700. The possible implications of this result for the polypeptide composition of the core reaction center are discussed.     

Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 μs in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f.

Incubation of chloroplasts with KCN or HgCl₂ decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700.

At low concentrations of salt and sugar the fast phases of P-700⁺ reduction were largely inhibited. Increasing concentrations of MgCl₂, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl₂ and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane.

The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin.

Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl₂ suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.     

The use of multi-frequency EPR techniques to identify the radicals produced in irradiated beta-blockers

The identification of radicals trapped in irradiated drugs can be very intricate. A multi-frequency electron paramagnetic resonance (EPR) study is proposed to resolve this problem. The Q-band (ca. 34 GHz) comparison with X-band (ca. 9 GHz) did not show significant differences for the four β-blockers studied (atenolol, esmolol, nadolol and propranolol). The use of a higher frequency (285 GHz) was required. It enabled us to determine the g-tensor values of the radicals present in atenolol and esmolol, respectively, g₁=2.0086, g₂=2.0059 and g₃=2.0021 and g₁=2.0066, g₂=2.0044 and g₃=2.0021. The latter was assigned as a phenoxyl radical, which can not be the case for the former. Therefore, radicals produced in esmolol may result from a more complex mechanism than the abstraction followed by the diffusion of an H atom inside the solid. In addition, two molecules as similar as atenolol and esmolol hydrochloride do not contain the same radicals after irradiation. These two conclusions drawn from the EPR results on β-blockers show clearly the importance of continuing the investigations on radiolytic mechanisms in solid-state drugs.     

Correlation of reaction-center chlorophyll (P-700) oxidation and bound iron-sulfur protein photoreduction in chloroplast Photosystem I at low temperatures

The extent of P-700 photooxidation at 18 °K has been followed in three different chloroplast preparations (unfractionated chloroplasts and two preparations enriched in Photosystem I). More than 90% of P-700⁺ formation in all preparations was eliminated by the addition of sodium dithionite at pH 10. Photoreduction of a bound chloroplast iron-sulfur protein was also decreased by at least 90% under similar conditions. Electron paramagnetic resonance spectra of the chloroplast preparations in the presence of dithionite showed chemical reduction of bound iron-sulfur protein under conditions where primary photochemistry is eliminated. These results indicate that P-700 photooxidation is concomitant with photoreduction of a bound iron-sulfur protein and that this iron-sulfur protein functions as the primary electron acceptor of Photosystem I.     

The orientation of the magnetic axes of membrane-bound iron-sulfur clusters and a cytochrome b-559 in the green halophilic alga Dunaliella parva

Photosynthetic membrane fragments separated from whole cells of the green alga Dunaliella parva, were oriented by incorporation into multilayers on thin Mylar films. These partially dehydrated films were then examined by EPR spectroscopy for evidence of orientation of paramagnetic components. Five previously identified paramagnetic components, the reduced states of iron-sulfur clusters A and B, the intermediate acceptor X^?, the reduced Rieske iron-sulfur cluster, and oxidized cytochrome b-559, displayed EPR signals showing orientation. In addition, several previously unknown paramagnetic components were also observed to be oriented. Four components, previously characterized in spinach chloroplast preparations, the iron-sulfur clusters A and B, the intermediate acceptor X^?, and cytochrome b-559, were shown to be similar in the green alga, D. parva. The orientations of iron-sulfur clusters A and B, however, were determined unambiguously in this preparation; this was not possible in previous work with spinach. The heme plane orientation of cytochrome b-559 was found to be perpendicular to the membrane plane in agreement with the results in spinach preparations. A new photoinduced EPR signal with g values of 1.88, 1.97 and 2.12 was seen only in the oriented preparations and was indicative of a reduced iron-sulfur cluster with an orientation different from that of iron-sulfur cluster A or B. This suggests the existence of a previously unidentified acceptor in Photosystem I of green plants. These studies clearly show that the orientation of these components in bioenergetic membranes are conserved over a large span of evolutionary development and are, therefore, an important aspect of the mechanism of electron transfer.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

Orientation of the hemes of high potential cytochromes relative to photosynthetic membranes, as shown by the linear dichroism of oriented preparations

The orientations of high potential cytochromes with respect to photosynthetic membranes was investigated in spinach chloroplasts and in Rhodopseudomonas viridis. The general approach consists in detection with polarized light of photoinduced absorbance changes related to the oxidation of the cytochromes. The orientation of cytochrome c-558 was measured at room temperature in chromatophores and whole cells of Rps. viridis, oriented on glass slides and in a magnetic field, respectively. The orientation of cytochrome b-559 of green plants was detected at 77 K in magnetically oriented chloroplasts. In both cases the dichroic ratio for the band shows that the heme plane makes an angle greater than 35°C with the membrane plane. Moreover, the dichroic ratio is not constant throughout the and β bands, for both cytochrome c-558 and b-559. Linear dichroism spectra of oriented pure horse heart cytochrome c and cytochrome c₂ of Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films show that the variations of the dichroic ratio in the and β bands can be explained by the occurrence of x- and y-polarized transitions absorbing at slightly different wavelengths.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.     

The inhibition of photosynthetic electron transport in spinach chloroplasts by low osmolarity.

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.     

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t_1/2 ≈ 120 μs. When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component (t_1/2 = 50–400 ms) reflecting the recombination between P-700⁺ and center X⁻ occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700⁺ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A⁻₁ as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

Location of the iron-sulfur clusters FA and FB in photosystem I: an electron paramagnetic resonance study of spin relaxation enhancement of P700+.

Photosystem I (PS I) mediates electron-transfer from plastocyanin to ferredoxin via a photochemically active chlorophyll dimer (P700), a monomeric chlorophyll electron acceptor (A0), a phylloquinone (A1), and three [4Fe-4S] clusters (FX/A/B). The sequence of electron-transfer events between the iron-sulfur cluster, FX, and ferredoxin is presently unclear. Owing to the presence of a 2-fold symmetry in the PsaC protein to which the iron-sulfur clusters F(A) and F(B) are bound, the spatial arrangement of these cofactors with respect to the C2-axis of symmetry in PS I is uncertain as well. An unequivocal determination of the spatial arrangement of the iron-sulfur clusters FA and FB within the protein is necessary to unravel the complete electron-transport chain in PS I. In the present study, we generate EPR signals from charge-separated spin pairs (P700+-FredX/A/B) in PS I and characterize them by progressive microwave power saturation measurements to determine the arrangement of the iron-sulfur clusters FX/A/B relative to P700. The microwave power at half saturation (P1/2) of P700+ is greater when both FA and FB are reduced in untreated PS I than when only FA is reduced in mercury-treated PS I. The experimental P1/2 values are compared to values calculated by using P700-FA/B crystallographic distances and assuming that either FA or FB is closer to P700+. On the basis of this comparison of experimental and theoretical values of spin relaxation enhancement effects on P700+ in P700+ [4Fe-4S]- charge-separated pairs, we find that iron-sulfur cluster FA is in closer proximity to P700 than the FB cluster.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.