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.     

Quantitative electron-paramagnetic-resonance measurements of the electron-transfer components of the photosystem-I reaction centre. The reaction-centre chlorophyll (P700), the primary electron acceptor X and bound iron-sulphur centre A.

An e.p.r. spectrum of the reduced form of the electron-transport component (X), thought to be the primary electron acceptor of Photosystem I, was obtained. By using line-shape simulations of this component and the free-radical e.p.r. signal I of the oxidized reaction-centre chlorophyll (P700), it was possible to determine the ratio of the number of electron spins to which these signals correspond in Photosystem-I particles under a variety of conditions. On illumination at cryogenic temperatures of Photosystem-I preparations, in which both bound iron-sulphur centres A and B were reduced, the measured ratio of free radical to component X varied between 1.04 and 2.23, with an average value of 1.54 +/- 0.18 where a Gaussian line-shape is assumed for the component-X signal in the simulation. The error in this measurement is estimated to be up to 50%. In a similar way component X and centre A of the bound iron-sulphur protein were quantified, the ratio between these two components varying between 1.26 and 0.61 with an average value of 0.75 +/- 0.06. These results indicate that the quantitative relationship, in terms of net electron spins, between centre A, component X and P700 is of the order to be expected if component X is indeed the primary electron acceptor in Photosystem I and a component of the photosynthetic electron-transport chain.     

Quantitative EPR studies of the primary reaction of Photosystem I in chloroplasts

Quantitative electron paramagnetic resonance studies of the primary event associated with Photosystem I in chloroplasts have been carried out at 25 °K. After illumination of either whole chloroplasts or Photosystem I subchloroplast fragments (D-144) with 715-nm actinic light at 25 °K, equal spin concentrations of oxidized P700 and reduced bound iron-sulfur protein (bound ferredoxin) have been measured. Quantitative determination of the concentration of these two carriers by EPR spectroscopy after illumination at low temperature indicates that Photosystem I fragments are enriched in P700 and the bound iron-sulfur protein as compared with unfractionated chloroplasts. These results indicate that P700 and the bound iron-sulfur protein function as the donor-acceptor complex of chloroplast Photosystem I.     

Absorption changes of P-700 reversible in milliseconds at low temperature in Triton-solubilized photosystem I particles.

Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 microseconds) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 +/- 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 +/- 30 microseconds) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2.6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 +/- 0.3 ms.     

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

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

Properties of the primary electron acceptor complex of photosystem I in the blue green alga Chlorogloea fritschii.

Photooxidation of P700 at low temperatures in membrane fractions from the blue-green alga $\text{Chlorogloea fritschii}$ may be coupled irreversibly to the reduction of a bound ferredoxin. If this ferredoxin is reduced before freezing, P700 photooxidation at low temperatures becomes reversible. This reversible photooxidation is coupled to the reduction of a component with an EPR signal at g = 2.08, 1.88 and 1.78. A complete spectrum of this component has been obtained for the first time. We propose that as in higher plants this component is the primary electron acceptor of Photosystem I, the bound ferredoxin is a secondary electron acceptor. Using ⁵⁷Fe enriched preparations we have shown that the ERP signals attributed to the bound ferredoxin are due to iron containing centres. This experiment did not show the presence of iron in the primary electron acceptor.     

Studies on P-700 photo-oxidation and reduction in Photosystem I subchloroplast particles

The photochemical oxidation and reduction of P-700 were studied in digitonin- and in sodium dodecyl sulphate (SDS)-Photosystem I (PS I) particles in the presence of ascorbate. In digitonin-PS I particles, reduction of P-700⁺ occurs by the bound iron-sulphur protein (P-430) and by ascorbate. The relative contribution of these back reactions depends on the length of the exposure to light and on the temperature and pH of the reaction medium. Experiments performed under anaerobic conditions demonstrate that some endogenous component may serve as the electron acceptor of P-430^?. The rate of the latter reaction is also dependent upon the temperature and pH of the sample. At pH 9 and lower temperatures the rate of this reaction is so much reduced that the reduction of P-700⁺ by ascorbate, which increases rapidly at high pH, can be observed even during illumination. The effects of secondary electron acceptors and of the presence of SDS on the absorption changes due to P-700 are also reported. Low concentrations of SDS are shown to retard the back reaction of P-700⁺ with P-430^?. Studies with SDS-PS I particles (CP_I) confirm the absence of the iron-sulphur centres in this preparation. Three larger P-700-chlorophylla-protein complexes prepared by mild electrophoresis in the presence of SDS plus Triton X-100, however, still contain P-430.     

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

Effect of temperature on the photoreduction of centres A and B in Photosystem I,and the kinetics of recombination

When spinach Photosystem I particles, frozen in the dark with ascorbate, are illuminated at low temperatures, one electron is transferred from P-700 to either iron-sulphur centre A or B. It was found that the proportion of centre A or B reduced depended on the temperature of illumination. At 25 K, reduction of centre A, as detected by ESR spectroscopy, was strongly preferred. At higher temperatures, at about 150K, there was an increased proportion of reduced centre B. Reduction of B was more strongly preferred in particles frozen in 50% glycerol. The kinetics of dark reoxidation of A^? and B^? at various temperatures were followed by observing the radical signal of P-700⁺, and also by periodically cooling to 25 K to measure the ESR spectra of the iron-sulphur centres. The recombination of A^? and P-700⁺ occurred at lower temperatures than that at of B^?; at 150–200 K, centre B was the more stable electron trap. Dark reoxidation of both centres was more rapid in samples that were illuminated at 25 K than in samples illuminated at 150–215 K. In no case was net electron transfer between centres A and B observed. Differences in g values of the ESR spectra in particles illuminated at 25 and 200 K indicate that the iron-sulphur centres are in altered conformational states. It is concluded firstly that, in the frozen state, the rates of dark electron transfer decrease in the sequence A^? → P-700⁺ > B^? → P-700⁺ > B^? → A; secondly, that when centres A or B are photoreduced, a temperature-dependent conformational change takes place which slows down the rate of recombination with P-700⁺.     

Absorption changes of P-700 reversible in milliseconds at low temperature in Triton-solubilized Photosystem I particles

Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 μs) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 ± 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 ± 30 μs) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2,6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 ± 0.3 ms.     

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.     

Comparison of the EPR properties of photosystem I iron-sulphur centres A and B in spinach and barley

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.     

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+ A2 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.     

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.     

Photochemical properties of a photosystem I subchloroplast fragment.

Treatment of spinach chloroplast fragments with the detergent lauryl dimethylamine oxide, followed by column chromatography on DEAE-cellulose, leads to the isolation of a Subchloroplast fragment that is enriched in Photosystem I. The spectrum of the lauryl dimethylamine oxide fragments, characterized by maxima at 418, 435, and 671 nm, shows the absence of chlorophyll b. The fragments contain 1 molecule of P700 per 40 chlorophyll molecules but have no cytochromes. The P700 in the fragments is photochemically active at both room temperature and liquid helium temperature. The fragments contain the primary electron acceptor of Photosystem I, as evidenced by the low-temperature photoreduction of a bound iron-sulfur protein. The fragments are able to catalyze noncyclic electron transfer from ascorbate to oxygen but not to the electron acceptor NADP.     

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-electrom 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 simular 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.     

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 that the low-potential (−700 mV) electron acceptor (X) in Photosystem I has two iron-sulphur centres

The Photosystem I reaction centre contains two groups of iron-sulphur centres: Fe-S_A and Fe-S_B with redox potentials between ?510 and ?590 mV, and Fe-S_X with redox potential about ?700 mV. Spin quantitation (Heathcote, P., Williams-Smith, D.L. and Evans, M.C.W. (1978) Biochem. J. 170, 373–378) and Mössbauer spectroscopy (Evans, E.H., Dickson, D.P.E., Johnson, C.E., Rush, J.D. and Evans, M.C.W. (1981) Eur. J. Biochem. 118, 81–84) did not show unequivocally whether Fe-S_X has one or two centres. Experiments are described which support the proposal that Fe-S_X has two centres. Fe-S_X can be photoreduced irreversibly by 210 K illumination of dithionite-reduced samples or reversibly by 7.5 K illumination of these samples. The amplitude of the Fe-S_X signal reversibly induced by illumination at 7.5 K is never more than 50% of the amplitude of the signal when Fe-S_X is prereduced by room temperature illumination or by 210 K illumination. Approx. half of the Fe-S_X is rapidly reduced by 210 K illumination, the remainder more slowly. The extent of reversible Fe-S_X reduction and P-700 photooxidation is little affected by the fast reduction of about half of the Fe-S_X. Subsequent reduction of the remaining Fe-S_X is paralleled by loss of the reversible photoreaction.     

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)).     

Radiation inactivation studies on Photosystem I. Functional sizes of electron-transport reactions

Radiation inactivation studies on the functional size of electron-transport processes in the Photosystem I reaction-centre complex showed the following characteristics. (1) The molecular mass required for electron transport from P-700 to iron-sulphur centre A was below 40 kDa. (2) Independent inactivation of iron-sulphur centres A and B was observed indicating their location on separate polypeptides. (3) The molecular mass of the polypeptides containing iron-sulphur centres A and B were 5–10 kDa based on a linear electron-transfer chain or 15–20 and 5–10 kDa (centre B) based on a branched chain. (4) A reaction centre ‘core’ containing the electron carriers for electron transport from P-700 to iron-sulphur centre X was indicated. These observations are discussed in comparison to current ideas on the polypeptide composition of the Photosystem I reaction centre. It is concluded that the radiation inactivation technique did not measure the size of Photosystem I polypeptides binding chlorophyll accounting for the small overall target size. The observed functional size came mostly from inactivation of the iron-sulphur centres showing that they are located on separate polypeptides.