Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X)

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).     

Assignment of a kinetic component to electron transfer between iron-sulfur clusters F(X) and F(A/B) of Photosystem I

We studied the kinetics of reoxidation of the phylloquinones in Chlamydomonas reinhardtii Photosystem I using site-directed mutations in the PhQ(A)-binding site and of the residues serving as the axial ligand to ec3(A) and ec3(B) chlorophylls. In wild type PS I, these kinetics are biphasic, and mutations in the binding region of PhQ(A) induced a specific slowing down of the slow component. This slowing allowed detection of a previously unobserved 180-ns phase having spectral characteristics that differ from electron transfer between phylloquinones and F(X). The new kinetic phase thus reflects a different reaction that we ascribe to oxidation of F(X)(-) by the F(A/B) FeS clusters. These absorption changes partly account for the differences between the spectra associated with the two kinetic components assigned to phylloquinone reoxidation. In the mutant in which the axial ligand to ec3(A) (PsaA-Met688) was targeted, about 25% of charge separations ended in P(700)(+)A(0)(-) charge recombination; no such recombination was detected in the B-side symmetric mutant. Despite significant changes in the amplitude of the components ascribed to phylloquinone reoxidation in the two mutants, the overall nanosecond absorption changes were similar to the wild type. This suggests that these absorption changes are similar for the two different phylloquinones and that part of the differences between the decay-associated spectra of the two components reflect a contribution from different electron acceptors, i.e. from an inter-FeS cluster electron transfer.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: The phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster FX

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A₁, the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F_X and the phylloquinone bound to either the PsaA (A_1A) or the PsaB (A_1B) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F_A and F_B. The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A₁) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F_X. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A_1B quinone and slightly endergonic, in the case of the A_1A quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A₀ on both electron transfer branches and the reduction of F_A by F_X.     

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.     

Control of electron transport in photosystem I by the iron-sulfur cluster FX in response to intra- and intersubunit interactions

Photosystem I (PS I) is a transmembranal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an approximately 10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I.     

The bound electron acceptors in green sulfur bacteria: resolution of the g-tensor for the F(X) iron-sulfur cluster in Chlorobium tepidum

The photosynthetic reaction center (RC) of green sulfur bacteria contains two [4Fe-4S] clusters named F(A) and F(B), by analogy with photosystem I (PS I). PS I also contains an interpolypeptide [4Fe-4S] cluster named F(X); however, spectroscopic evidence for an analogous iron-sulfur cluster in green sulfur bacteria remains equivocal. To minimize oxidative damage to the iron-sulfur clusters, we studied the sensitivity of F(A) and F(B) to molecular oxygen in whole cells of Chlorobium vibrioforme and Chlorobium tepidum and obtained highly photoactive membranes and RCs from Cb. tepidum by adjusting isolation conditions to maximize the amplitude of the F(A)(-)/F(B)(-) electron paramagnetic resonance signal at g = 1.89 (measured at 126 mW of microwave power and 14 K) relative to the P840(+) signal at g = 2.0028 (measured at 800 microW of microwave power and 14 K). In these optimized preparations we were able to differentiate F(X)(-) from F(A)(-)/F(B)(-) by their different relaxation properties. At temperatures between 4 and 9 K, isolated membranes and RCs of Cb. tepidum show a broad peak at g = 2.12 and a prominent high-field trough at g = 1.76 (measured at 126 mW of microwave power). The complete g-tensor of F(X)(-), extracted by numerical simulation, yields principal values of 2.17, 1.92, and 1. 77 and is similar to F(X) in PS I. An important difference from PS I is that because the bound cytochrome is available as a fast electron donor in Chlorobium, it is not necessary to prereduce F(A) and F(B) to photoaccumulate F(X)(-).     

Rapid evolutionary divergence of Photosystem I core subunits PsaA and PsaB in the marine prokaryote Prochlorococcus

The nucleotide sequences of the genes coding for the subunits of the Photosystem I (PS I) core, PsaA and PsaB were determined for the marine prokaryotic oxyphototrophs Prochlorococcus sp. MED4 (CCMP1378), P. marinus SS120 (CCMP1375) and Synechococcus sp. WH7803. Divergence of these sequences from those of both freshwater cyanobacteria and higher plants was remarkably high, given the conserved nature of PsaA and PsaB proteins. In particular, the PsaA of marine prokaryotes showed several specific insertions and deletions with regard to known PsaA sequences. Even in between the two Prochlorococcus strains, which correspond to two genetically different ecotypes with shifted growth irradiance optima, the sequence identity was only 80.2% for PsaA and 88.9% for PsaB. Possible causes and implications of the fast evolution rates of these two PS I core subunits are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase

We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.     

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.     

Stabilization of Photosystem II (O(2) Evolution) of Spinach Chloroplasts by Radiation-induced Immobilization

Spinach chloroplasts were immobilized with vinyl monomers by radiation-induced polymerization at low temperature and stored in buffer containing bovine serum albumin. The lifetime of O₂ evolution activity in photosystem II was prolonged remarkably in immobilized chloroplasts. Thermostability of immobilized chloroplasts stored in buffer containing bovine serum albumin was far better than that of immobilized chloroplasts in pure buffer and that of intact chloroplasts. When immobilized chloroplasts were stored in buffer including polyethylene glycol, the lifetime of O₂ evolution activity was longer than for those stored in buffer containing bovine serum albumin.     

A possible role for iron-sulfur cluster N2 in proton translocation by the NADH: ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The enzyme mechanism is still unknown due to the lack of a high-resolution structure and its complicated composition. The complex from Escherichia coli is made up of 13 subunits called NuoA through NuoN and contains one FMN and nine iron-sulfur (Fe/S) clusters as redox groups. The pH dependence of the midpoint redox potential of the Fe/S cluster named N2 and its spin-spin interaction with ubiquinone radicals made it an ideal candidate for a key component in redox-driven proton translocation. During the past years we have assigned the subunit localization of cluster N2 to subunit NuoB by site-directed mutagenesis and predicted its ligation by molecular simulation. Redox-induced FT-IR spectroscopy has shown that its redox reaction is accompanied by the protonation and deprotonation of individual amino acid residues. These residues have been identified by site-directed mutagenesis. The enzyme catalytic activity depends on the presence of cluster N2 and is coupled with major conformational changes. From these data a model for redox-induced conformation-driven proton translocation has been derived.     

Friedreich's ataxia variants I154F and W155R diminish frataxin-based activation of the iron-sulfur cluster assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ～15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ～ I154F > W155F > W155A ～ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.     

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.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.     

ESR signal of the iron-sulfur center F(X) and its function in the homodimeric reaction center of Heliobacterium modesticaldum

Electron transfer in the membranes and the type I reaction center (RC) core protein complex isolated from Heliobacterium modesticaldum was studied by optical and ESR spectroscopy. The RC is a homodimer of PshA proteins. In the isolated membranes, illumination at 14 K led to accumulation of a stable ESR signal of the reduced iron-sulfur center F(B)(-) in the presence of dithiothreitol, and an additional 20 min illumination at 230 K induced the spin-interacting F(A)(-)/F(B)(-) signal at 14 K. During illumination at 5 K in the presence of dithionite, we detected a new transient signal with the following values: g(z)= 2.040, g(y)= 1.911, and g(x)= 1.896. The signal decayed rapidly with a 10 ms time constant after the flash excitation at 5 K and was attributed to the F(X)(-)-type center, although the signal shape was more symmetrical than that of F(X)(-) in photosystem I. In the purified RC core protein, laser excitation induced the absorption change of a special pair, P800. The flash-induced P800(+) signal recovered with a fast 2-5 ms time constant below 150 K, suggesting charge recombination with F(X)(-). Partial destruction of the RC core protein complex by a brief exposure to air increased the level of the P800(+)A(0)(-) state that gave a lifetime (t(1/2)) of 100 ns at 77 K. The reactions of F(X) and quinone were discussed on the basis of the three-dimensional structural model of RC that predicts the conserved F(X)-binding site and the quinone-binding site, which is more hydrophilic than that in the photosystem I RC.     

A kinetic assessment of the sequence of electron transfer from F(X) to F(A) and further to F(B) in photosystem I: the value of the equilibrium constant between F(X) and F(A)

The x-ray structure analysis of photosystem I (PS I) crystals at 4-A resolution (Schubert et al., 1997, J. Mol. Biol. 272:741-769) has revealed the distances between the three iron-sulfur clusters, labeled F(X), F(1), and F(2), which function on the acceptor side of PS I. There is a general consensus concerning the assignment of the F(X) cluster, which is bound to the PsaA and PsaB polypeptides that constitute the PS I core heterodimer. However, the correspondence between the acceptors labeled F(1) and F(2) on the electron density map and the F(A) and F(B) clusters defined by electron paramagnetic resonance (EPR) spectroscopy remains controversial. Two recent studies (Diaz-Quintana et al., 1998, Biochemistry. 37:3429-3439;, Vassiliev et al., 1998, Biophys. J. 74:2029-2035) provided evidence that F(A) is the cluster proximal to F(X), and F(B) is the cluster that donates electrons to ferredoxin. In this work, we provide a kinetic argument to support this assignment by estimating the rates of electron transfer between the iron-sulfur clusters F(X), F(A), and F(B). The experimentally determined kinetics of P700(+) dark relaxation in PS I complexes (both F(A) and F(B) are present), HgCl(2)-treated PS I complexes (devoid of F(B)), and P700-F(X) cores (devoid of both F(A) and F(B)) from Synechococcus sp. PCC 6301 are compared with the expected dependencies on the rate of electron transfer, based on the x-ray distances between the cofactors. The analysis, which takes into consideration the asymmetrical position of iron-sulfur clusters F(1) and F(2) relative to F(X), supports the F(X) --> F(A) --> F(B) --> Fd sequence of electron transfer on the acceptor side of PS I. Based on this sequence of electron transfer and on the observed kinetics of P700(+) reduction and F(X)(-) oxidation, we estimate the equilibrium constant of electron transfer between F(X) and F(A) at room temperature to be approximately 47. The value of this equilibrium constant is discussed in the context of the midpoint potentials of F(X) and F(A), as determined by low-temperature EPR spectroscopy.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

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.     

The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.