Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center ‘X’ and correlation with optical signal ‘A2’

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700⁺ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center ‘X’, characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700⁺ A⁻₂ reaction pair and the ESR P-700⁺ center X⁻ reaction pair to indicate that signal A₂ and center X represent the same iron-sulfur center in Photosystem I.     

Electron spin resonance spectrum of species "X" which may function as the primary electron acceptor in photosystem I of green plant photosynthesis.

Purified Photosystem I particles from spinach when reduced with 10 mM dithionite at pH 9 exhibited a 50% light reversible-ESR Signall (P-700+) at about 10 K. It was possible to show by signal-averaging techniques that a light-reversible ESR spectrum concomitant with the reversible Single 1 can be observed with approximate principal g factors at g = 2.07, g = 1.86 and g = 1.75.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

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.     

Primary photochemistry of Photosystem I in chloroplasts. Dynamics of reversible charge separation in open reaction centers at 25 K

The reduction rate of oxidized reaction center chlorophyll of Photosystem I after laser-flash excitation at 25 K has been determined for D-144 subchloroplast fragments and chloroplasts. A maximum of 40% of Photosystem I reaction centers undergo irreversible charge separation (P-700, Cluster A: P-700⁺, Cluster A^?) at 25 K, a percentage which is independent of laser-flash intensity. The remaining reaction centers in chloroplasts and D-144 fragments undergo reversible charge separation with biphasic recombination. Similar amplitudes and time constants (chloroplasts, 49 μs (61%); D-144 fragments, 90 μs (67%)) were obtained for the fast component, while the slower component differed considerably in time (chloroplasts, 2.9 ms; D-144 fragments, 170 ms). It is known that Fe-S Cluster A is photoreduced in less than 1 ms at 25 K. Data obtained support a model for Photosystem I involving a single intermediate in the decay path between the reduced primary electron acceptor (A^?₁) and P-700⁺ and a second intermediate in the decay path between a reduced secondary electron acceptor and P-700⁺. Dual laser-flash experiments to determine rate constants for these processes are included.     

Electron spin resonance spectrum of species “X” which may function as the primary electron acceptor in Photosystem I of green plant photosynthesis

Purified Photosystem I particles from spinach when reduced with 10 mM dithionite at pH 9 exhibited a 50% light reversible-ESR Signal 1 (P-700⁺) at about 10 K. It was possible to show by signal-averaging techniques that a light-reversible ESR spectrum concomitant with the reversible Signal 1 can be observed with approximate principal g factors at g = 2.07, g = 1.86 and g = 1.75.     

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I. II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77-84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with [D] = 279 X 10(-4) cm-1 and [E] = 39 X 10(-4) cm-1. The triplet resonances titrate with a midpoint potential of +380 +/- 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and delta Hpp = 1.0 mT. Deconvolution of the P-700+ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species A0, which is characterized by g = 2.0033 +/- 0.0004 and delta Hpp = 1.0 +/- 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A1 in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A1 is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A1 in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A1 from its binding site.     

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 less than or equal to T less than or equal to 100 K. T1 was 200 mus at 100 K and increased to 900 mus at 10 K. T2 was 40 ns at 40 K and increased to 100 ns at 10 K. T1 for 40 K less than or equal to T less than or equal to 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 P-700+ signal. The absence of dipolar 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.     

PRoperties of the low-temperature photosystem I primary reaction in the P-700-chlorophyll alpha-protein.

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15 degrees K in P-700-chlorophyll alpha-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300 degrees K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15 degrees K as well as at 300 degrees K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.     

Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Flash photolysis-electron spin resonance studies of photosystem I. A fast reduction of component of P-700+.

A 300 mus decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 mus actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375 +/- 10 mV (pH = 7.5). These data suggest the assignment of the 300-mus decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171-1174 (1971)).     

Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Flash photolysis electron spin resonance studies of the electron acceptor species at low temperatures in photosystem I of spinach subchloroplast particles.

The light-induced electron spin resonance signals of Photosystem I spinach subchloroplast particles have been studied at approximately 6 degrees K. Using the technique of flash photolysis-electron spin resonance with actinic illumination at 647 nm, a kinetic analysis of the previously observed bound ferredoxin ESR signals was carried out. Signal I (P700+) exhibits a partial light-reversible behavior at 6 degrees K so it was expected that if the bound ferredoxin is the primary acceptor of Photosystem I, it should also exhibit a partial reversible behavior. However, none of the bound ferredoxin ESR signals showed any such light reversible behavior. A search to wider fields revealed two components which did exhibit the expected kinetic behavior. These components are very broad (about 80 G) and are centered at g equals to 1.75 and g equals to 2.07. These two components exhibit the expected characteristics of the primary electron acceptor. A model is presented to account for the reversible and irreversible photochemical changes in Photosystem I. The possible identity of the primary acceptor responsible for these two new components, is discussed in terms of the available information. The primary acceptor may be an iron-sulfur protein, but not of the type characteristic of the bound or water-soluble ferredoxins found so far in chloroplasts.     

Photosystem I charge separation in the absence of center A and B. III. Biochemical characterization of a reaction center particle containing P-700 and FX

The Photosystem I reaction center is a membrane-bound, multiprotein complex containing a primary electron donor (P-700), a primary electron acceptor (A₀), an intermediate electron acceptor (A₁) and three membrane-bound iron-sulfur centers (F_X, F_B, and F_A). We reported in part I of this series (Golbeck, J.H. and Cornelius, J.M. (1986) Biochim. Biophys. Acta 849, 16–24) that in the presence of 1% lithium dodecyl sulfate (LDS), the reaction center becomes dissociated, resulting in charge separation and recombination between P-700 and F_X without the need for prereduction of F_A and F_B. In this paper, we report (i) the LDS-induced onset of the 1.2-ms ‘fast’ phase of the P-700 absorption transient is time-dependent, attaining a maximum 3:1 ratio of ‘fast’ to ‘slow’ kinetic phases; (ii) the ‘fast’ kinetic phase, corresponding to the P-700⁺ F_X⁻ backreaction, is stabilized indefinitely by dilution of the LDS-treated particle followed by ultrafiltration over a YM-100 membrane; (iii) without stabilization, the P-700⁺ F_X⁻ reaction deteriorates, leading to the rise of the long-lived P-700 triplet formed from the P-700⁺A_O⁻ backreaction; (iv) the ‘slow’ kinetic phase correlates with the redox and ESR properties of F_A and/or F_B, which indicates that in a minority of particles the terminal iron-sulfur protein remains attached to the reaction center core; (v) the ultrafiltered reaction center is severely deficient in all of the low molecular-weight polypeptides, particularly the 19-kDa, 18-kDa and 12-kDa polypeptides relative to the 64-kDa polypeptide(s); (vi) the stabilized particle contains 5.8 mol labile sulfide per mol photoactive P-700, reflecting largely the iron-sulfur content of F_x, but also residual F_A and F_B, on the reaction center; and (vii) the apoproteins of F_A and F_B are physically removed from the reaction center particle as indicated by the presence of protein-bound zero-valence sulfur in the YM-100 filtrate. These results are interpreted in terms of a model for Photosystem I in which F_A and F_B are located on a low-molecular-weight polypeptide and F_X is depicted as a [2Fe-2S] cluster shared between the two high-molecular-weight polypeptides Photosystem I-A1 and Photosystem I-A2.     

Heat-Stability of Iron-Sulfur Centers and P-700 in Photosystem I Reaction Center Complexes Isolated from the Thermophilic Cyanobacterium Synechococcus elongatus

Stabilities of iron-sulfur centers and reaction center chlorophyllP-700 in Photosystem I reaction center complex (CP1-a), isolatedby sodium dodecyl sulfate treatment from the thermophilic cyanobacteriumSynechococcus elongatus, were studied by EPR and optical spectroscopy.P-700 was destroyed by treatment at temperatures above 80?Cfor 5 minutes with a half inactivation temperature of 93?C.The three iron-sulfur centers F_A, F_B and F_X showed similar thermalstabilities and were half inactivated at about 70?C. Thus, theisolated Photosystem I reaction center complexes of S. elongatusare still highly resistant to heat. (Received May 9, 1990; Accepted June 25, 1990)     

Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Effects on the binding activity with receptor sites and interactions between subunits.

The properties of the component 'X' identified as the primary electron acceptor of Photosystem I in spinach was investigated by electron-paramagnetic-resonance spectroscopy and the complete spectrum obtained for the first time. Component 'X' has gx = 1.78, gy = 1.88 and gz = 2.08; it can be observed only at very low temperatures (8--13K) and high microwave powers. Component X was identified in Photosystem I particles prepared with the French press or with Triton X-100. In samples reduced with ascorbate, illumination at low temperatures results in the photo-oxidation of P700 and reduction of a bound iron-sulphur protein; this is irreversible at low temperature. In samples in which the iron-sulphur proteins are reduced by sodium dithionite, illumination at low temperature results in the oxidation of P700 and the reduction of component 'X'; this is reversible at low temperature. The light-induced P700 signal is the same size with either ascorbate or dithionite as reducing agent, showing that all of the P700 involved in reduction of bound ferredoxin also functions in the reduction of component 'X'.     

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 (P-700) and X. Oxidation-reduction potential titrations confirmed that Centre A had Em congruent to -550 mV, Centre B had Em congruent to -585 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, P-700 photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible P-700 photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with Em congruent to -585 mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.     

A flash photolysis ESR study of photosystem II signal IIvf, the physiological donor to P-680+.

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (g approximately 2.0045, deltaHpp approximately 19G). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with "red" illumination (lambda less than 680 nm) and occurs stoichiometrically in a 1:1 ratio with P-700+. The Photosystem II transient is formed in less than 100 mus and decays via first-order kinetics with a halftime of 400-900 mus. Additionally, the t1/2 for radical decay is temperature independent between 20 and 4 degrees C; however, below 4 degrees C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal-mol-1. Inhibition of electron transport through Photosystem II by o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1-5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this "quinoidal" type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, P-680+.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts.

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 degrees K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth approximately 8 gauss) which decays with a half-time of 5.0 ms at 35 degrees K. The half-time of decay is independent of temperature in the range of 10-77 degrees K. This reversible signal can be eliminated by preillumination of the sample at 35 degrees K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to - 150 mV prior to freezing. In the presence of ferricyanide (20-50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 degrees K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680+ and the primary electron acceptor.     

Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S^2? →S⁰ conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.