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     

Spectroscopic evidence for the presence of an iron-sulfur center similar to Fx of Photosystem I inHeliobacillus mobilis

Treatment of membranes ofHeliobacillus mobilis with high concentrations of the chaotropic agent urea resulted in the removal of the iron-sulfur centers F_A and F_B from the reaction center, as indicated by EPR spectra under strongly reducing conditions. In urea-treated membranes, transient absorption measurements upon a laser flash indicated a recombination between the photo-oxidized primary donor P798⁺ and a reduced acceptor with a time constant of 20 ms at room temperature. Benzylviologen, vitamin K-3 and methylene blue were found to accept electrons from the reduced acceptor efficiently. A differential extinction coefficient of 225–240 mM^–1 cm^–1 at 798 nm was determined from experiments in the presence of methylene blue. Transient absorption difference spectra between 400 and 500 nm in the presence and absence of artificial acceptors indicated that the electron acceptor involved in the 20 ms recombination has an absorption spectrum similar to that of an iron-sulfur center. This iron-sulfur center was assigned to be analogous to F_X of Photosystem I. Our results provide evidence in support of the presence of F_X in heliobacteria, which was proposed on the basis of the reaction center polypeptide sequence (Liebl et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7124–7128). Implications for the electron transfer pathway in the reaction center of heliobacteria are discussed.     

Excitation energy transfer and charge separation in the isolated Photosystem II reaction center

The nature of excitation energy transfer and charge separation in isolated Photosystem II reaction centers is an area of considerable interest and controversy. Excitation energy transfer from accessory chlorophyll a to the primary electron donor P680 takes place in tens of picoseconds, although there is some evidence that thermal equilibration of the excitation between P680 and a subset of the accessory chlorophyll a occurs on a 100-fs timescale. The intrinsic rate for charge separation at low temperature is accepted to be ca. (2 ps)^–1, and is based on several measurements using different experimental techniques. This rate is in good agreement with estimates based on larger sized particles, and is similar to the rate observed with bacterial reaction centers. However, near room temperature there is considerable disagreement as to the observed rate for charge separation, with several experiments pointing to a ca. (3 ps)^–1 rate, and others to a ca. (20 ps)^-1 rate. These processes and the experiments used to measure them will be reviewed.Abbreviations Chl chlorophyll - FWHM full-width at half-maximum - Pheo pheophytin - PS II Photosystem II - P680 primary electron donor of the Photosystem II reaction center - RC reaction center The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

A ferredoxin-type iron-sulfur protein gene,frx B,is expressed in the chloroplasts of tobacco and spinach

Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the frxB gene identified from DNA sequence analysis.     

Reconstitution of iron-sulfur center FB results in complete restoration of NADP+ photoreduction in Hg-treated Photosystem I complexes from Synechococcus sp. PCC 6301

The F_B iron-sulfur cluster is destroyed preferentially by treating Photosystem I complexes with HgCl₂(Kojima Y, Niinomi Y, Tsuboi S, Hiyama T and Sakurai H (1987) Bot Mag 100: 243–53). When F_B is 95% depleted but F_Ais quantitatively retained in cyanobacterial PS I complexes, the reduction potential of F_A remains highly electronegative (E_m=–530 mV, n=1), the EPR spectral and spin relaxation properties of F_A and F_Xremain unchanged, but NADP⁺ photoreduction rates decline from 552 to 72 mol mg Chl^–1 h^–1.When F_B is reconstituted with FeCl₃, Na₂S and -mercaptoethanol, NADP⁺photoreduction rates recover to 528 mol mg Chl^–1 h^–1. The correlation between the presence of F_Band NADP⁺ photoreduction provides direct experimental evidence that this iron-sulfur cluster is required for electron throughput from cytochromec ₆ to flavodoxin or ferredoxin in Photosystem I.Abbreviations Chl chlorophyll - DPIP dichlorophenolindophenol - PS I Photosystem I Published as Journal Series #11091 of the University of Nebraska Agricultural Research Division. This paper is dedicated to the memory of the late Professor Daniel Arnon, who is remembered for his gracious and generous encouragement of the senior author's early career.     

Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec ₅₅₁ encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of F_A and F_B of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.     

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.     

Structure of cyanobacterial Photosystem I

Photosystem I is one of the most fascinating membrane protein complexes for which a structure has been determined. It functions as a bio-solar energy converter, catalyzing one of the first steps of oxygenic photosynthesis. It captures the light of the sun by means of a large antenna system, consisting of chlorophylls and carotenoids, and transfers the energy to the center of the complex, driving the transmembrane electron transfer from plastoquinone to ferredoxin. Cyanobacterial Photosystem I is a trimer consisting of 36 proteins to which 381 cofactors are non-covalently attached. This review discusses the complex function of Photosystem I based on the structure of the complex at 2.5 Å resolution as well as spectroscopic and biochemical data.     

Diazonium modification of Photosystem I. A specific effect on iron-sulfur Center B

Modification of chloroplast membranes with diazonium benzene sulfonate (DABS) leads to a loss of Photosystem I-dependent ferredoxin reduction but not methyl viologen reduction. EPR studies of DABS-modified membranes show no inhibition of P-700⁺ formation at cryogenic temperatures, but iron-sulfur Center A photoreduction is markedly inhibited. Iron-sulfur Center B photoreduction at physiological temperatures in DABS-modified membranes is also markedly inhibited and little Center B can be detected after dark chemical reduction. These results indicate DABS specifically modifies iron-sulfur Center B of the spinach chloroplast Photosystem I electron acceptor complex and that Center B is obligately required for the reduction of Center A at cryogenic temperatures. Possible electron transport pathways at physiological temperatures are also considered.     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

Structure-function studies on the interaction of PsaC with the Photosystem 1 heterodimer

The interaction of PsaC with the core heterodimer of Photosystem 1 was studied in wild type Synechocystis sp. PCC 6803 and the site-directed mutant R561E of PsaB. The mutant reaction center was much less stable in urea and the functional reconstitution of the mutant core using PsaC was impaired. However, the extent of reconstitution increased in the presence of divalent cations whereas that of the wild type was inhibited. We conclude that the reaction center in the mutant is unstable, most likely due to the introduction of an unfavorable electrostatic interaction between surface-exposed residues on PsaC and the binding site for the subunit on the core heterodimer in support of the model proposed previously (Rodday et al. (1993) Photosynth Res 36: 1–9).Abbreviations PEG polyethylene glycol - 1 - PS1 Photosystem 1 - PMSF phenylmethylsulphonyl fluorid - TMPD N, N, N, N tetramethyl-p-phenylenediamine - TPCK N-tosyl-Phe chloromethyl ketone     

Short-term responses of Photosystem I to heat stress

When 23°C-grown potato leaves (Solanum tuberosum L.) were exposed for 15 min to elevated temperatures in weak light, a dramatic and preferential inactivation of Photosystem (PS) II was observed at temperatures higher than about 38°C. In vivo photoacoustic measurements indicated that, concomitantly with the loss of PS II activity, heat stress induced a marked gas-uptake activity both in far-red light (>715 nm) exciting only PS I and in broadband light (350–600 nm) exciting PS I and PS II. In view of its suppression by nitrogen gas and oxygen and its stimulation by high carbon-dioxide concentrations, the bulk of the photoacoustically measured gas uptake by heat-stressed leaves was ascribed to rapid carbon-dioxide solubilization in response to light-modulated stroma alkalization coupled to PS I-driven electron transport. Heat-induced gas uptake was observed to be insensitive to the PS II inhibitor diuron, sensitive to the plastocyanin inhibitor HgCl₂ and saturated at a rather high photon flux density of around 1200 E m^–2 s^–1. Upon transition from far-red light to darkness, the oxidized reaction center P700⁺ of PS I was re-reduced very slowly in control leaves (with a half time t_1/2 higher than 500 ms), as measured by leaf absorbance changes at around 820 nm. Heat stress caused a spectacular acceleration of the postillumination P700⁺ reduction, with t_1/2 falling to a value lower than 50 ms (after leaf exposure to 48°C). The decreased t_1/2 was sensitive to HgCl₂ and insensitive to diuron, methyl viologen (an electron acceptor of PS I competing with the endogenous acceptor ferredoxin) and anaerobiosis. This acceleration of the P700⁺ reduction was very rapidly induced by heat treatment (within less than 5 min) and persisted even after prolonged irradiation of the leaves with far-red light. After heat stress, the plastoquinone pool exhibited reduction in darkness as indicated by the increase in the apparent Fo level of chlorophyll fluorescence which could be quenched by far-red light. Application (for 1 min) of far-red light to heat-pretreated leaves also induced a reversible quenching of the maximal fluorescence level Fm, suggesting formation of a pH gradient in far-red light. Taken together, the presented data indicate that PS I responded to the heat-induced loss of PS II photochemical activity by catalyzing an electron flow from stromal reductants. Heat-stress-induced PS I electron transport independent of PS II seems to constitute a protective mechanism since block of this electron pathway in anaerobiosis was observed to result in a dramatic photoinactivation of PS I.Abbreviations PFD photon flux density - PS Photosystem - Apt and Aox amplitude of the photothermal and photobaric components of the photoacoustic signal, respectively - P700 reaction center pigment of PS I - Fo and Fm initial and maximal levels of chlorophyll fluorescence, respectively - Fv=Fm Fo-variable chlorophyll fluorescence - Q_A primary (stable) electron acceptor of PS II - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Cyt cytochrome     

Electron Fluxes through Photosystem I in Cucumber Leaf Discs Probed by far-red Light

Cucumber leaf discs were illuminated at room-temperature with far-red light to photo-oxidise P700, the chlorophyll dimer in Photosystem (PS) I. The post-illumination kinetics of P700(+) re-reduction were studied in the presence of inhibitors or cofactors of photosynthetic electron transport. The re-reduction kinetics of P700(+) were well fitted as the sum of three exponentials, each with its amplitude and rate coefficient, and an initial flux (at the instant of turning off far-red light) given as the product of the two. Each initial flux is assumed equal to a steady state flux during far-red illumination. The fast phase of re-reduction, with rate coefficient k (1) approximately 10 s(-1), was completely abolished by a saturating concentration of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); it is attributed to electron flow to P700(+) from PS II, which was stimulated to some extent by far-red light. The intermediate phase, with rate coefficient k (1) approximately 1 s(-1), was only partly diminished by methyl viologen (MV) which diverts electron flow to oxygen. The intermediate phase is attributed to electron donation from reduced ferredoxin to the intersystem pool; reduced ferredoxin could be formed: (1) directly by electron donation on the acceptor of PS I; and/or (2) indirectly by stromal reductants, in line with only a partial inhibition of the intermediate phase by MV. Duroquinol enhanced the intermediate phase in the presence of DCMU, presumably through its interaction with thylakoid membrane components leading to the partial reduction of plastoquinone. The slow phase of P700(+) re-reduction, with rate coefficient k (1) approximately 0.1 s(-1), was unaffected by DCMU and only slightly affected by MV; it could be associated with electron donation to either: (1) the intersystem chain by stromal reductants catalysed by NAD(P)H dehydrogenase slowly; or (2) plastocyanin/P700(+) by ascorbate diffusing across the thylakoid membrane to the lumen. It is concluded that a post-illumination analysis of the fluxes to P700(+) can be used to probe the pathways of electron flow to PS I in steady state illumination.     

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.     

Purification and crystallization of Photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002

An active photosystem (PSI) complex was isolated from a phycobilisome-less mutant of the mesophilic cyanobacterium Synechococcus PCC 7002 by a mild procedure. Purification of PS I was achieved using a sucrose density gradient and an isoelectric focussing subsequent to the extraction of PSI from thylakoids with dodecyl--maltoside. Electron microscopy and gel filtration HPLC suggested that the isolated complex represents a trimeric form of PSI. The trimeric form was resistant to pH or detergent exchange. A molecular weight of 690 kDa to 760 kDa has been determined for the complex by gel filtration HPLC in several detergents or mixtures of detergents.The PSI complex contains the polypeptides of the psaA, psaB, psaC, psaD, psaE, psaL gene products and two small polypeptides as determined by SDS-PAGE and N-terminal sequencing; its antenna size is 77±2 Chl a/P₇₀₀. The full set of Fe-S clusters (F_A, F_B and F_X) was observed by EPR-spectroscopy. A preliminary characterization of crystals obtained from this preparation was carried out using SDS-PAGE, optical and EPR spectroscopy.Abbreviations BA benzamidine - CAS 6-amino-n-caproic acid - C₈-G octyl--D-glucopyranoside - C₁₂-M lauryl--D-maltoside - C₁₀-M decyl--D-maltoside - C₈-TG octyl--D-thioglucoside - Chl a chlorophyll a - EPR electron paramagnetic resonance - F_A, F_B, F_X iron-sulfur centers - HPLC high perfomance liquid chromatography - kDa kilodalton(s) - LDAO lauryldimethylamine oxide - MES 2-(N-morpholino)ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - P₇₀₀ primary electron donor - SB12 sulfobetain 12 - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tris tris(hydroxymethyl)-aminomethane     

Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction

The variation of the rate of cyclic electron transport around Photosystem I (PS I) during photosynthetic induction was investigated by illuminating dark-adapted spinach leaf discs with red + far-red actinic light for a varied duration, followed by abruptly turning off the light. The post-illumination re-reduction kinetics of P700⁺, the oxidized form of the photoactive chlorophyll of the reaction centre of PS I (normalized to the total P700 content), was well described by the sum of three negative exponential terms. The analysis gave a light-induced total electron flux from which the linear electron flux through PS II and PS I could be subtracted, yielding a cyclic electron flux. Our results show that the cyclic electron flux was small in the very early phase of photosynthetic induction, rose to a maximum at about 30 s of illumination, and declined subsequently to <10% of the total electron flux in the steady state. Further, this cyclic electron flow, largely responsible for the fast and intermediate exponential decays, was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, suggesting an important role of redox poising of the cyclic components for optimal function. Significantly, our results demonstrate that analysis of the post-illumination re-reduction kinetics of P700⁺ allows the quantification of the cyclic electron flux in intact leaves by a relatively straightforward method.     

Spectral and kinetic characterization of electron acceptor A1 in a Photosystem I core devoid of iron-sulfur centers FX,FB and FA

The kinetic and spectroscopic properties of the secondary electron acceptor A₁ were determined by flash absorption spectroscopy at room and cryogenic temperatures in a Photosystem I (PS I) core devoid of the iron-sulfur clusters F_X, F_B and F_A. It was shown earlier (Warren, P.V., Golbeck, J.H. and Warden, J.T. (1993) Biochemistry 32: 849–857) that the majority of the flash-induced absorbance increase at 820 nm, reflecting formation of P700⁺, decays with a t_1/2 of 10 s due to charge recombination between P700⁺ and A₁ ^–. Following A₁ ^– directly around 380 nm, where absorbance changes due to the formation of P700⁺ are negligible, two major decay components were resolved in this study with t_1/2 of 10 s and 110 s at an amplitude ratio of 2.5:1. The difference spectra between 340 and 490 nm of the two kinetic phases are highly similar, showing absorbance increases from 340 to 400 nm characteristic of the one-electron reduction of the phylloquinone A₁. When measured at 10 K, the flash-induced absorbance changes around 380 nm can be fitted with two decay phases of t_1/2 15 s and 150 s at an amplitude ratio 1:1. The difference spectra of both kinetic phases from 340 to 400 nm are similar to those determined at 298 K and are therefore attributed to charge recombination in the pair P700⁺A₁ ^–. These results indicate that the backreaction between P700⁺ and A₁ ^– is multiphasic when F_X, F_B and F_A are removed, and only slightly temperature dependent in the range of 298 K to 10 K.Abbreviations Chl chlorophyll - D pathlength for the measuring light through the sample - DPIP 2,6-dichlorophenolindophenol - EPR electron paramagnetic resonance - IR infrared - PS I Photosystem I - Tris Tris(hydroxymethyl)aminomethane - UV ultraviolet Published as Journal Series #10890 of the University of Nebraska Agricultural Research Division and supported by a grant from the National Science Foundation (MCB-9205756).     

Effect of the Pool Size of Stromal Reductants on the Alternative Pathway of Electron Transfer to Photosystem I in Chloroplasts of Intact Leaves

The effect of elevated temperature on electron flow to plastoquinone pool and to PSI from sources alternative to PSII was studied in barley (Hordeum vulgare L.) and maize (Zea mays L.) leaves. Alternative electron flow was characterized by measuring variable fluorescence of chlorophyll and absorption changes at 830 nm that reflect redox changes of P700, the primary electron donor of PSI. The treatment of leaves with elevated temperature resulted in a transient increase in variable fluorescence after cessation of actinic light. This increase was absent in leaves treated with methyl viologen (MV). The kinetics of P700⁺ reduction in barley and maize leaves treated with DCMU and MV exhibited two exponential components. The rate of both components markedly increased with temperature of the heat pretreatment of leaves when the reduction of P700⁺ was measured after short (1 s) illumination of leaves. The acceleration of both kinetic components of P700⁺ reduction by high-temperature treatment was much less pronounced when P700⁺ reduction rate was measured after illumination of leaves for 1 min. Since the treatment of leaves with DCMU and MV inhibited both the electron flow to PSI from PSII and ferredoxin-dependent cycling of electrons around PSI, the accelerated reduction of P700⁺ indicated that high temperature treatment activated electron flow to PSII from reductants localized in the chloroplast stroma. We conclude that the lesser extent of activation of this process by elevated temperature after prolonged illumination of heat-inhibited leaves is caused by depletion of the pool stromal reductants in light due to photoinduced electron transfer from these reductants to oxygen.     

Heterogeneity of Photosystem I reaction centers in barley leaves as related to the donation from stromal reductants

The light-response curves of P700 oxidation and time-resolved kinetics of P700⁺ dark re-reduction were studied in barley leaves using absorbance changes at 820 nm. Leaves were exposed to 45 °C and treated with either diuron or diuron plus methyl viologen (MV) to prevent linear electron flow from PS II to PSI and ferredoxin-dependent cyclic electron flow around PSI. Under those conditions, P700⁺ could accept electrons solely from soluble stromal reductants. P700 was oxidized under weak far-red light in leaves treated with diuron plus MV, while identical illumination was nearly ineffective in diuron-treated leaves in the absence of MV. When heat-exposed leaves were briefly illuminated with strong far-red light, which completely oxidized P700, the kinetics of P700⁺ dark reduction was fitted by a single exponential term with half-time of about 40 ms. However, two first-order kinetic components of electron flow to P700⁺ (fast and slow) were found after prolonged leaf irradiation. The light-induced modulation of the kinetics of P700⁺ dark reduction was reversed following dark adaptation. The fast component (half time of 80–90 ms) was 1.5 larger than the slow one (half time of about 1 s). No kinetic competition occurred between two pathways of electron donation to P700⁺ from stromal reductants. This suggests the presence of two different populations of PSI. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fluorescence detected magnetic resonance of the primary donor and inner core antenna chlorophyll in Photosystem I reaction centre protein: Sign inversion and energy transfer

The Photosystem I reaction centre protein CP1, isolated from barley using polyacrylamide gel electrophoresis showed an EPR (Electron Paramgnetic Resonance) spectrum with the polarisation pattern AEEAAE, typical of the primary donor triplet state ³P700, created via radical pair formation and recombination. ³P700 could also be detected by Fluorescence Detected Magnetic Resonance (FDMR) at _f > 700 nm even in the presence of a large number of chlorophyll antennae. Its zero field splitting parameters, D=282.5×10^-4 cm^-1 and E=38.5×10^-4 cm^-1, were independent of the detection wavelength, and agreed with ADMR (Absorption Detected Magnetic Resonance) and EPR values. The signs of the ³P700 D+E and D-E transitions were positive (increase in fluorescence intensity on applying a resonance microwave field). In contrast, in the emission band 685 < _f < 700 nm FDMR spectra with negative D+E and D-E transitions were detected, and the D value was wavelength-dependent. These FDMR results support an excitation energy transfer model for CP1, derived from time-resolved fluorescence studies, in which two chlorophyll antenna forms are distinguished, with fluorescence at 685 < _f < 700 nm (inner core antennae, F690), and _f > 700 nm (low energy antenna sites, F720), in addition to the P700. The FDMR spectrum in F690 emission can be interpreted as that of ³P700, observed via reverse singlet excitation energy transfer and added to the FDMR spectrum of the antenna triplet states generated via intramolecular intersystem crossing. This would indicate that reversible energy transfer between F690 and P700 occurs even at 4.2 K.Abbreviations Chl chlorophyll - CP1 core chlorophyll protein of Photosystem I - EPR electron paramagnetic resonance - F690, F720 chlorophyll forms having fluorescence maximum at 690–695 and 720 nm, respectively - F(A)(O)DMR fluorescence (absorption) (optical) detected magnetic resonance - FF fluorescence fading - ISC intramolecular intersystem crossing - _f fluorescence emission wave-length - LHC I light harvesting chlorophyll a/b protein of Photosystem I - P700 primary donor of Photosystem I - PS I Photosystem I - RC reaction centre - RP radical pair - SDS sodium dodecyl sulphate - ZFS zero field splitting