The Interaction of Water-Soluble Nitroxide Radicals with Photosystem II.

In this work, we investigated the redox transients of a number of water-soluble spin labels upon their interactions with Photosystem II (PS II) core complexes isolated from spinach leaves. We have found that the reactivity of nitroxide radicals, determined by the rate of their reduction upon illumination of PS II, depends on the chemical structure of radicals and the capability of their coming close to low-potential redox centers of photoactive PS II complexes. An enhanced capability of nitroxide radicals to accept electrons from PS II correlates with their chemical structure. Nitroxide radicals NTI (2,2,5,5-tetramethyl-4-nitromethylene-3-imidazolidine-N-oxyl) and Tacet (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl-acetate), containing polar groups, appear to be most efficient acceptors of electrons donated by PS II compared to neutral (TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) or positively charged (Tamine, 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl) spin labels. We assume that enhanced reactivities of polar nitroxide radicals, NTI and Tacet, are determined (1) by their relatively high redox potentials, providing the possibility to accept electrons from PS II, and (2) by their affinities to the closest binding sites on the surface of PS II in the vicinity of the primary plastoquinone acceptor PQA (12-14 Å) or/and in the intraprotein cavity for the secondary plastoquinone PQB (~ 22 Å).

Hydroxyectoine protects Mn-depleted photosystem II against photoinhibition acting as a source of electrons.

In the present study, we have investigated the effect of hydroxyectoine (Ect-OH), a heterocyclic amino acid, on oxygen evolution in photosystem II (PS II) membrane fragments and on photoinhibition of Mn-depleted PS II (apo-WOC-PS II) preparations. The degree of photoinhibition of apo-WOC-PS II preparations was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to restore the amplitude of light-induced changes of chlorophyll fluorescence yield (∆F). It was found that Ect-OH (i) stimulates the oxygen-evolving activity of PS II, (ii) accelerates the electron transfer from exogenous electron donors (K4[Fe(CN)6], DPC, TMPD, Fe2+, and Mn2+) to the reaction center of apo-WOC-PS II, (iii) enhances the protective effect of exogenous electron donors against donor-side photoinhibition of apo-WOC-PS II preparations. It is assumed that Ect-OH can serve as an artificial electron donor for apo-WOC-PS II, which does not directly interact with either the donor or acceptor side of the reaction center. We suggest that the protein conformation in the presence of Ect-OH, which affects the extent of hydration, becomes favorable for accepting electrons from exogenous donors. To our knowledge, this is the first study dealing with redox activity of Ect-OH towards photosynthetic pigment-protein complexes.

[Treatment of patients with lower limb varicose veins disease]. / Lechenie patsientov s varikoznoi bolezn'iu nizhnikh konechnostei.

Modern data on the prevalence and pathophysiology of lower limb varicose disease are presented. The results of studies of modern approaches to the surgical correction of this state are demonstrated. Conclusions about the unresolved number of problems.

Electron Transfer through the Acceptor Side of Photosystem I: Interaction with Exogenous Acceptors and Molecular Oxygen.

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin-Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A1A/A1B in the symmetric redox cofactor branches of PS I and iron-sulfur clusters FX, FA, and FB have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.

Formation and decay of P680 (P(D1)-P(D2))⁺PheoD1⁻ radical ion pair in photosystem II core complexes.

Under physiological conditions (278 K) femtosecond pump-probe laser spectroscopy with 20-fs time resolution was applied to study primary charge separation in spinach photosystem II (PSII) core complexes excited at 710 nm. It was shown that initial formation of anion radical band of pheophytin molecule (Pheo⁻) at 460 nm is observed with rise time of ~11ps. The kinetics of the observed rise was ascribed to charge separation between Chl (chlorophyll a) dimer, primary electron donor in PSII (P680*) and Pheo located in D1 protein subunit (PheoD1) absorbing at 420 nm, 545 nm and 680 nm with formation of the ion-radical pair P680⁺PheoDI⁻. The subsequent electron transfer from Pheo(D1)⁻ to primary plastoquinone electron acceptor (Q(A)) was accompanied by relaxation of the 460-nm band and occurred within ~250 ps in good agreement with previous measurements in Photosystem II-enriched particles and bacterial reaction centers. The subtraction of the P680⁺ spectrum measured at 455 ps delay from the spectra at 23 ps or 44 ps delay reveals the spectrum of Pheo(DI)⁻, which is very similar to that measured earlier by accumulation method. The spectrum of Pheo(DI)⁻ formation includes a bleaching (or red shift) of the 670 nm band indicating that Chl-670 is close to Pheo(D1). According to previous measurements in the femtosecond-picosecond time range this Chl-670 was ascribed to Chl(D1) [Shelaev, Gostev, Vishnev, Shkuropatov, Ptushenko, Mamedov, Sarkisov, Nadtochenko, Semenov and Shuvalov, J. Photochemistry and Photobiology, B: Biology 104 (2011) 45-50]. Stimulated emission at 685 nm was found to have two decaying components with time constants of ~1ps and ~14ps. These components appear to reflect formation of P680⁺Chl(D1)⁻ and P680⁺Pheo(D1)⁻, respectively, as found earlier. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Electron transfer in photosystem I containing native and modified quinone acceptors.

The pigment-protein complex of photosystem I (PS I) catalyzes light-driven oxidation of plastocyanin or cytochrome c6 and reduction of ferredoxin or flavodoxin in oxygenic photosynthetic organisms. In this review, we describe the current state of knowledge of the processes of excitation energy transfer and formation of the primary and secondary ion-radical pairs within PS I. The electron transfer reaction involving quinone cofactor in the A1 site and its role in providing asymmetry of electron transport as well as interaction with oxygen and ascorbate in PS I are discussed.

Effect of trehalose on oxygen evolution and electron transfer in photosystem 2 complexes.

The pigment-protein complex of photosystem 2 (PS 2) catalyzes the light-driven oxidation of water molecule and the reduction of plastoquinone. In this work, we studied the effect of the disaccharide trehalose, which is unique in its physicochemical properties, on isolated PS 2 complex. It was found that trehalose significantly stimulated the steady-state rate of oxygen evolution. The study of single flash-induced fluorescence decay kinetics demonstrated that trehalose did not affect the rate of QA(-) oxidation, although it led to an increase in the relative fractions of PS 2 reaction centers capable of QA(-) oxidation. Trehalose also prevented PS 2 complexes from being inactivated on prolonged storage. We propose that in the presence of trehalose, which affects the extent of hydration, the protein can preferentially exist in a more optimal conformation for effective functioning.

Mechanism of primary and secondary ion-radical pair formation in photosystem I complexes.

The mechanisms of the ultrafast charge separation in reaction centers of photosystem I (PS I) complexes are discussed. A kinetic model of the primary reactions in PS I complexes is presented. The model takes into account previously calculated values of redox potentials of cofactors, reorganization energies of the primary P700(+)A0(-) and secondary P700(+)A1(-) ion-radical pairs formation, and the possibility of electron transfer via both symmetric branches A and B of redox-cofactors. The model assumes that the primary electron acceptor A0 in PS I is represented by a dimer of chlorophyll molecules Chl2A/Chl3A and Chl2B/Chl3B in branches A and B of the cofactors. The characteristic times of formation of P700(+)A0(-) and P700(+)A1(-) calculated on the basis of the model are close to the experimental values obtained by pump-probe femtosecond absorption spectroscopy. It is demonstrated that a small difference in the values of redox potentials between the primary electron acceptors A0A and A0B in branches A and B leads to asymmetry of the electron transfer in a ratio of 70 : 30 in favor of branch A. The secondary charge separation is thermodynamically irreversible in the submicrosecond range and is accompanied by additional increase in asymmetry between the branches of cofactors of PS I.

Primary radical ion pairs in photosystem II core complexes.

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700-710 nm at 278 K. It was found that the initial charge separation between P680* and ChlD1 (Chl-670) takes place with a time constant of ~1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680*((1-δ)) (P680(δ+)ChlD1(δ-)), where δ ~ 0.5. The subsequent electron transfer from P680(δ+)ChlD1(δ-) to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (ChlD1(δ-)) and bleaching of the PheoD1 bands at 420, 545, and 680 nm with development of the Pheo(-) band at 460 nm. Further electron transfer to QA occurs within 250 ps in accordance with earlier data. The spectra of P680(+) and Pheo(-) formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ~1 and ~13 ps due to the formation of P680(δ+)ChlD1(δ-) and P680(+)PheoD1(-), respectively.

Vectorial charge transfer reactions on the donor side of manganese-depleted and reconstituted photosystem 2 core complexes.

The light-induced functioning of photosystem 2 (PS 2) is directly linked to the translocation of both electrons and protons across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). Generation of ΔΨ due to S-state transitions of the water oxidation complex was demonstrated for the first time in Mn-depleted and reconstituted PS 2 core complexes incorporated into liposomes. The kinetics and relative amplitudes of the electrogenic reactions in dark-adapted samples during S1→S2, S2→S3, and S4→S0 transitions in response to the first, second and third laser flashes were comparable to those obtained in the intact PS 2 core particles. These results expand current understanding of the nature and mechanisms of electrogenic (vectorial) reactions due to a charge transfer on the donor side of PS 2.

Transmembrane electric potential difference in the protein-pigment complex of photosystem 2.

The protein-pigment complex of photosystem 2 (PS2) localized in the thylakoid membranes of higher plants, algae, and cyanobacteria is the main source of oxygen on Earth. The light-induced functioning of PS2 is directly linked to electron and proton transfer across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). The major contribution to ΔΨ of the PS2 reaction center is due to charge separation between the primary chlorophyll donor P(680) and the quinone acceptor Q(A), accompanied by re-reduction of P(680)(+) by the redox-active tyrosine residue Y(Z). The processes associated with the uptake and release of protons on the acceptor and donor sides of the enzyme, respectively, are also coupled with ΔΨ generation. The objective of this work was to describe the mechanisms of ΔΨ generation associated with the S-state transitions of the water-oxidizing complex in intact PS2 complex and in PS2 preparation depleted of Mn(4)Ca cluster in the presence of artificial electron donors. The findings elucidate the mechanisms of electrogenic reactions on the PS2 donor side and may be a basis for development of an effective solar energy conversion system.

Primary steps of electron and energy transfer in photosystem I: effect of excitation pulse wavelength.

Time-resolved differential spectra of photosystem I complex were obtained by the "pump-probe" technique with 25-fs pulses with maxima at 670, 700, and 720 nm. The ratio between the number of excited chlorophyll molecules of the antenna and of the reaction center was shown to depend on spectral characteristics of the pump pulses. In all cases, an ultrafast (<150 fs) formation of the primary radical pair P700(+)A(0)() was recorded. However, on excitation by pulses with maxima at 670 or 700 nm, detection of the charge separation was masked by the much more intensive bleaching at the chlorophyll Q(y) band due to excitation of the bulk antenna chlorophylls. We show that triggering the charge separation by 25-fs pulses centered at 720 nm allows to detect more clearly kinetics of formation of the primary and secondary ion-radical pairs. The findings help to explain possible reasons for discrepancies of kinetics of primary steps of electron transfer detected in different laboratories.

Electron transfer between exogenous electron donors and reaction center of photosystem 2.

Transfer of electrons between artificial electron donors diphenylcarbazide (DPC) and hydroxylamine (NH2OH) and reaction center of manganese-depleted photosystem 2 (PS2) complexes was studied using the direct electrometrical method. For the first time it was shown that reduction of redox-active amino acid tyrosine Y(Z)(.) by DPC is coupled with generation of transmembrane electric potential difference (DeltaPsi). The amplitude of this phase comprised ~17% of that of the DeltaPsi phase due to electron transfer between Y(Z) and the primary quinone acceptor Q(A). This phase is associated with vectorial intraprotein electron transfer between the DPC binding site on the protein-water interface and the tyrosine Y(Z)(.). The slowing of DeltaPsi decay in the presence of NH2OH indicates effective electron transfer between the artificial electron donor and reaction center of PS2. It is suggested that NH2OH is able to diffuse through channels with diameter of 2.0-3.0 A visible in PS2 structure and leading from the protein-water interface to the Mn(4)Ca cluster binding site with the concomitant electron donation to Y(Z)(.). Because the dielectrically-weighted distance between the NH2OH binding site and Y(Z)(.) is not determined, the transfer of electrons from NH2OH to Y(Z)(.) could be either electrically silent or contribute negligibly to the observed electrogenicity in comparison with hydrophobic donors.

Manganese-depleted/reconstituted photosystem II core complexes in solution and liposomes.

Chlorophyll fluorescence transients measurements were employed to study the functioning of spinach photosystem II (PS II) core complexes in solution or reconstituted into liposomes. Lipid vesicles were prepared from soybean phospholipids (asolectine) or a mixture of spinach thylakoid lipids. In comparison with intact PS II core complexes comprising two distinct fluorescence phases, designated as O-J and J-P, complete suppression of the latter phase in Mn-depleted samples was observed. An increase of magnitude of the J-P phase in the presence of exogenous MnCl(2) (4 Mn/RC) indicate in favor of partial restoring of oxygen-evolution activity of PS II. The J-P phase observed in PS II in solution was characterized by a lifetime of ~320 ms, while in liposome-reconstituted samples this phase was accelerated up to ~20 ms in case of asolectine and up to ~9 ms in case of a mixture of thylakoid lipids. These data clearly suggest that lipid environment stimulates the steady-state rate of oxygen evolution. The effect of lipids is likely based on keeping the embedded proteins in optimal structure for efficient functioning.

P680 (P(D1)P(D2)) and Chl(D1) as alternative electron donors in photosystem II core complexes and isolated reaction centers.

Low temperature (77-90 K) measurements of absorption spectral changes induced by red light illumination in isolated photosystem II (PSII) reaction centers (RCs, D1/D2/Cyt b559 complex) with different external acceptors and in PSII core complexes have shown that two different electron donors can alternatively function in PSII: chlorophyll (Chl) dimer P(680) absorbing at 684 nm and Chl monomer Chl(D1) absorbing at 674 nm. Under physiological conditions (278 K) transient absorption difference spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach PSII core complexes excited at 710 nm. It was shown that the initial electron transfer reaction takes place with a time constant of ~0.9 ps. This kinetics was ascribed to charge separation between P(680)* and Chl(D1) absorbing at 670 nm accompanied by the formation of the primary charge-separated state P(680)(+)Chl(DI)(-), as indicated by 0.9-ps transient bleaching at 670 nm. The subsequent electron transfer from Chl(D1)(-) occurred within 13-14 ps and was accompanied by relaxation of the 670-nm band, bleaching of the Pheo(D1) Q(x) absorption band at 545 nm, and development of the anion-radical band of Pheo(D1)(-) at 450-460 nm, the latter two attributable to formation of the secondary radical pair P(680)(+)Pheo(D1)(-). The 14-ps relaxation of the 670-nm band was previously assigned to the Chl(D1) absorption in isolated PSII RCs [Shelaev, Gostev, Nadtochenko, Shkuropatov, Zabelin, Mamedov, Semenov, Sarkisov and Shuvalov, Photosynth. Res. 98 (2008) 95-103]. We suggest that the longer wavelength position of P(680) (near 680 nm) as a primary electron donor and the shorter wavelength position of Chl(D1) (near 670 nm) as a primary acceptor within the Q(y) transitions in RC allow an effective competition with an energy transfer and stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as the primary electron donor and Pheo(D1) as the primary acceptor cannot be ruled out, the 20-fs excitation at the far-red tail of the PSII core complex absorption spectrum at 710 nm appears to induce a transition to a low-energy state P(680)* with charge-transfer character (probably P(D1)(δ+)P(D2)(δ-)) which results in an effective electron transfer from P(680)* (the primary electron donor) to Chl(D1) as the intermediary acceptor.

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex.

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.

Voltage changes involving photosystem II quinone-iron complex turnover.

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q (A) (-) and the oxidized non-heme iron Fe(3+) on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Deltapsi (negative charging of the proteoliposome interior) indicates that the iron-quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (tau approximately 0.1 micro s) of Deltapsi generation related to electron transfer between the redox-active tyrosine Y(Z) and Q(A), an additional phase in the submillisecond time domain (tau approximately 0.1 ms at 23 degrees C, pH 7.0) and relative amplitude approximately 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q(A) and the secondary quinone Q(B). The rate of the additional electrogenic phase is decreased by about one-half in the presence of D(2)O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.

Electrogenic protonation of the secondary quinone acceptor Q(B) in spinach photosystem II complexes incorporated into lipid vesicles.

The generation of transmembrane electric potential difference (delta psi) in quinone acceptor complex of proteoliposomes containing core complexes of photosystem II from spinach was studied using for the measurements a direct electrometric technique. Besides the fast increase in the membrane potential associated with the electron transfer between the redox-active tyrosine 161 residue (Y(Z)) in D1 polypeptide and the primary quinone acceptor Q(A), an additional electrogenic phase with tau approximately 0.85 msec at pH 7.3 and the maximal relative amplitude of approximately 11% of the Y(Z)ox Q(A)- phase was observed after the second light flash. The sensitivity of this phase to diuron (an inhibitor of electron transfer between Q(A) and the secondary quinone acceptor Q(B)), the dependence of its amplitude on the light flash parity, and also a decrease in its rate constant with increase in pH indicated that it was due to dismutation of Q(A)- and Q(B)- with the subsequent protonation of a doubly reduced plastoquinone molecule: Q(A)- Q(B)- + 2H+ --> Q(A)Q(B)H2.

Dielectric and photoelectric properties of photosynthetic reaction centers.

A brief review of studies of dielectric and photoelectric properties of photosynthetic reaction centers of purple bacteria as well as photosystem I and photosystem II of cyanobacteria and higher plants is given. A simple kinetic model of the primary processes of electron transfer in photosynthesis is used to discuss possible mechanisms of correlation between rate constant of charge transfer reaction, free energy of electron transition, and effective dielectric constant in the locus of corresponding carriers.