Transfer of light-induced electron-spin polarization from the intermediary acceptor to the prereduced primary acceptor in the reaction center of photosynthetic bacteria

In reaction centers and chromatophores of photosynthetic bacteria strong light-induced emissive ESR signals have been found, not only after a flash, but also under continuous illumination. The signal, with g = 2.0048 and ΔH_pp = 7.6 G, is only present under reducing conditions in material in which the primary acceptor, ubiquinone, U and its associated high-spin ferrous ion are magnetically uncoupled. Its amplitude under continuous illumination is strongly dependent on light intensity and on microwave power.

The emissive signal is attributed to the prereduced primary acceptor, U⁻, which becomes polarized through transfer of spin polarization by a magnetic exchange interaction with the photoreduced, spin polarized intermediary acceptor, I⁻. A kinetic model is presented which explains the observed dependence of emissivity on light intensity and microwave power. Applying this analysis to the light saturation data, a value of the exchange rate between I⁻ and U⁻ of 4 · 10⁸ s⁻¹ is derived, corresponding to an exchange interaction of 3–5 G.     

Electron spin polarization (CIDEP) of a primary electron acceptor in Photosystem II

Photosystem II particles prepared according to Berthold et al. (Berthold, D.A., Babcock, G.T. and Yocum. C.F. (1981) FEBS Lett. 134, 231–234) and to Ganago and Klimov (Ganago, I.B. and Klimov, V.V. (1985) Biofizika, in the press) were subjected to an iron extraction procedure and cooled in the light under reducing conditions. The samples showed a 0.9 mT wide EPR line at g = 2.0044 attributed to the reduced primary acceptor Q⁻_A. Further prolonged illumination at 15 K generated a wide, somewhat asymmetric EPR signal at g = 2.0034−2.0038 that showed strong, reversible polarization upon continuous illumination at 15 K and below. The signal is ascribed to an acceptor that becomes spin-polarized through exchange-mediated transfer of polarization as described previously for photosynthetic bacteria (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 502–535). Arguments are given that the aceptor may be intermediate between the pheophytin transient acceptor and Q_A.     

A new EPR signal attributed to the primary plastosemiquinone acceptor in Photosystem II

A study of signals, light-induced at 77 K in O₂-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q^?_AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin^? signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin^? signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.     

Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides.

The photoreductive trapping of the transient, intermediate acceptor, I-, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I- was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 400, 587--599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301--304). The optical changes of I- showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I- were observed: one was a narrow singlet at g = 2.0035, deltaH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I- on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI-Q-Fe2+ and the singlet EPR signal is due to DI-Q2-Fe2+. : formula: (see text), where D is the primary donor (BChl)2+. The above model was supported by the following observations: (1) During the first illumination, sigmoidal kinetics of the formation of I- was observed. This is a direct consequence of the three-sequential reactions. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I-. This is due to the dark decay, k4, to the state DIQ2-Fe2+ formed after the first illumination. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k1, is operative. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I-(Q-Fe2%) in reaction centers containing menaquinone. The value of tau2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone (T = 20 degrees C). The temperature and pH dependence of the dark electron transfer reaction I-(Q-Fe2+) yields I(Q2-Fe2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I- to MQ-Fe2+ using Hopfield's theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10(-3) eV. From this value the distance between I- and MQ- was estimated to be 7.5--10 A.     

Magnetic interactions between the triplet state of the primary donor and the prereduced ubiquinone acceptor in reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1

The lineshape of the polarized prereduced primary quinone acceptor of Rhodopseudomonas sphaeroides after a laser flash was studied using time-resolved continuous wave EPR and electron spin echo techniques. The EPR as well as the electron spin echo experiments show that the lineshape of the ubiquinone shortly after a laser flash is time-dependent. This is attributed to magnetic interactions between the ubiquinone and the triplet state of the primary donor. Taking into account both dipolar and exchange interactions, a simulation of the EPR lineshape of the ubiquinone at 50 μs after the laser flash is performed, yielding a distance of 1.85 nm between the primary donor and the primary acceptor. From the simulation it is also concluded that the plane of the ubiquinone is approximately parallel to the surface of the membrane.     

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 °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 °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 = 1.75 and g = 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.     

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

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

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.     

EPR and optical spectroscopic properties of the electron carrier intermediate between the reaction center bacteriochlorophylls and the primary acceptor in Chromatium vinosum.

1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c553 and c555 oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c553 is oxidized irreversibly in milliseconds by light at 7 K. 2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c553 and the reaction center bacteriochlorophyll dimer (BChl)2 absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c553 hemes are equivalent with respect to the light induced (BChl)2+. At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity. 3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow (t1/2 approximately equal to 30 s) oxidation of one cytochrome c553 heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinone-iron. The state (oxidized c553/reduced I) relaxes in the dark at 200K in t1/2 approx. 20 min but below 77 K it is trapped on a days time scale. 4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I-. is consistent with the idea that the reduced intermediary carrier is not simply BPh-. but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state...     

Anomalous acceleration of the photocycle in photosynthetic reaction centers inhibited on the acceptor side

The rate of the photocycle (quinone reduction cycle) was measured under continuous light excitation in an isolated reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides. The rate is determined by the slowest step of the photocycle, which could be the photochemistry (charge separation), the quinone/quinol and cytochrome c(2+)/c(3+) exchanges, or proton delivery to the secondary quinone. The photocycle was driven by high light intensity of a laser diode (5 W/cm(2) at 808 nm) to avoid light limitation of the observed rate. The fast turnover of the reaction center (up to 10(3) s(-1)) was slowed down by inhibition of the proton delivery to the secondary quinone by transition metal ions (Cd(2+) and Ni(2+)), by mutation of a key protonatable group (L213Asp --> Asn), or by use of low-affinity ubiquinone (UQ(0)) to the secondary quinone binding site. Although in all of these cases the rate of turnover was 2-3 orders of magnitude less than that of the primary photochemistry, marked light intensity dependence was observed. The rate of the photocycle increased from 7 s(-1) (Ni(2+), low light intensity) to 27 s(-1) (high light intensity) at pH 8.4. The anomalous reacceleration is due to alternative events on the acceptor side induced by continuous excitation. We argue that the continuous excitation of the protein trapped in the reduced acceptor (Q(A)(-)Q(B)(-)) state produces short-lived reduced bacteriopheophytin (I(-)) that delivers activation energy to anomalous changes on the acceptor side as second interquinone electron transfer before proton uptake or increase of the quinone dissociation constant.     

Characterization of the primary radical pair in reaction centers of Heliobacillus mobilis by 13C photo-CIDNP MAS NMR

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by (13)C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the (13)C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 8(1)-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.     

Turnover of ubiquinone-0 at the acceptor side of photosynthetic reaction center

The steady-state operation of photosynthetic reaction center from Rhodobacter sphaeroides was investigated by measuring the rate of cytochrome photo-oxidation under intensive continuous illumination (808 nm, 5 W cm(-2)). The native quinone UQ(10) in Q(B) binding site of the reaction center was substituted by tailless UQ(0) and the binding parameters and the turnover rate of the UQ(0) was studied to test the recently discovered light-intensity dependent acceptor side effect (Gerencsér and Maróti 2006). The binding parameters of UQ(0) (k (on) = 2.1 x 10(5) M(-1) s(-1) and k (off) = 100 s(-1)) were characteristic to the RC exposed to high light-intensity. The dissociation constant (K (D) = 480 muM) determined under high light intensity is 2-3 times larger than that determined from flash-experiments. The light-intensity dependent acceleration of cytochrome turnover measured on reaction center of inhibited proton binding was independent of the type of the quinone and was sensitive only to the size ("pressure") of the quinone pool. The dissociation constants of different types of semiquinones show similarly high (several orders of magnitude) increase in the modified conformation of the Q(B) binding pocket due to high intensity of illumination. This result indicates the exclusive role of the quinone headgroup in the binding of semiquinone to different conformations of the protein.     

Evidence for an anisotropic magnetic interaction between the (bacteriopheophytin) intermediary acceptor and the first quinone acceptor in bacterial photosynthesis

We have investigated electron spin polarization effects occurring in protonated and perdeuterated reaction centers of Rhodospirillum rubrum with electron spin resonance at 9 and 35 GHz (X- and Q-band). As for Rhodopseudomonas sphaeroides strains 2.4.1 and R-26 (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 520–535; Gast, P., Mushlin, R.A. and Hoff, A.J. (1982) J. Phys. Chem. 86, 2886–2891), electron spin polarization effects of the prereduced first quinone acceptor Q^?_A in R. rubrum are strongly nonuniform. This nonuniformity is due to an anisotropic magnetic coupling between the intermediary bacteriopheophytin acceptor (I^?) and Q^?_A. It is argued that the anisotropy is too strong to arise solely from an anisotropy in the exchange interaction between I^? and Q^?_A and that dipolar contributions to the magnetic coupling between I^? and Q^?_A are important. The anisotropy in the magnetic coupling for reaction centers of Rps. sphaeroides strains 2.4.1 and R-26 is different from that of R. rubrum wild type. The combination of the 4-fold higher resolution at Q-band and the line narrowing upon deuteration has enabled us to obtain the principal g values and two hyperfine interaction constants of the reduced first quinone acceptor Q^?_A. The principal g values are g_x = 2.0067, g_y = 2.0056 and g_z = 2.0024; the hyperfine constant of the CH₂ group at position 1 is 1.6 G and that of the CH₃ group at position 2 is 2.1 G. These values are close to those found for ubisemiquinone in vitro (Okamura, M.Y., Debus, R.J., Isaacson, R.A. and Feher, G. (1980) Fed. Proc. 39, 1802; Hales, B.J. (1975) J. Am. Chem. Soc. 97, 5993–5997).     

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex.

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.     

EPR studies by Fe isotopic substitution on the nature of an unknown electron acceptor in Azotobacter vinelandii phosphorylating particles

1. EPR ⁵⁷Fe isotopic substitution studies provide unequivocal evidence that the g = 2.011 signal found in oxidized Azotobacter vinelandii phosphorylating particles is due to an iron-containing structure. The broadening constant determined as a result of this electron—nuclear hyperfine interaction was 15.7 G.

2. A similar signal found in a number of iron—sulfur containing proteins was found by quantitative EPR estimations to exist in a variable but substantial concentration when compared to the intensity of the reduced g = 1.9 type EPR resonance.

3. Reaction of the phosphorylating particles with excess potassium ferricyanide resulted in an alteration of the initial g = 2.011 iron signal resulting in the detection by microwave power studies of at least two different iron species which exhibited major g-values at 1.992 and 2.027.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II_slow and Signal II_fast of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y_Z and Y_D of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043 ± 0.0003 and a line width of 10 ± 1 G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q_A in the absence of magnetic interaction with the non-heme Fe²⁺. We conclude that peroxynitrite impairs PSII electron transport in the Q_AFe²⁺ niche.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

Functions of pheophytin, plastoquinone, iron and carotenoids in plant photosystem 2 reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Ph), in photosystem-2 (PS-2) reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and Eh approximately -450 mV produces a narrow ESR signal of Ph. (g = 2.0033; delta H approximately 13 G) and a "doublet" centered at g = 2.00 with a splitting of 52 G at 7 K. The doublet (but not the narrow signal) is eliminated after extraction of lyophylized TSF-II with hexane, containing 0.1-0.2% methanol, or after extraction of Fe with LiClO4 and o-phenantroline, and the signal is restored by reconstitution with plastoquinone-A (PQ) or Fe++, respectively. The Fe removal results also in the development of a photoinduced ESR signal of PQ. (g approximately 2.0044; delta H approximately 9.2 G). The conclusion is made that the primary electron acceptor, Q, is in fact a complex PQ-Fe++ and that the exchange interaction of Ph. with PQ. -Fe++ is responsible for the ESR doublet. Photoreduction of Ph in TSF-IIa is accompanied by the 3-fold decrease in the formation of carotenoid triplet state (measured by the characteristic flash-induced absorbance changes) which is suggested to be a result of charge recombination in the pair [P680+ .PH.].     

The Effect of Oxygen at Physiological Levels on the Detection of free Radical Intermediates by Electron Paramagnetic Resonance

It is well known that oxygen enhances Che relaxation of free radical EPR probes through spin lattice and Heisenberg spin-spin interactions with consequent effect on the line height and width. The two relaxation processes have opposing effects on the signal heights and depend on the concentration of oxygen, the incident microwave power, and the presence of other paramagnetic species. During EPR studies of chemical, biochemical, and cellular processes involving free radicals, molecular oxygen has significant magnetic influence on the EPR signal intensity of the free radical species under investigation in addition to affecting the rates of production of the primary species and the stability of the spin adduct nitroxides. These effects are often overlooked and can cause artifacts and lead to erroneous interpretation. In the present study, the effects of oxygen and ferricyanide on the EPR signal height of stable and persistent spin adduct nitroxides at commonly employed microwave powers were examined. The results show that under commonly adopted EPR spectrometer instrumental conditions, artifactual changes in the EPR signal of spin adducts occur and the best way to avoid them is by keeping the oxygen level constant using a gas-permeable cell.