Ivan I. Proskuryakov

Production of superoxide in chloroplast thylakoid membranes ESR study with cyclic hydroxylamines of different lipophilicity.

Accumulation of nitroxide radicals, DCP or TMT , under illumination of a thylakoid suspension containing either hydrophilic, DCP‐H, or lipophilic, TMT‐H, cyclic hydroxylamines that have high rate constants of the reaction with superoxide radicals, was measured using ESR. A slower accumulation of TMT in contrast with DCP accumulation was explained by re‐reduction of TMT by the carriers of the photosynthetic electron transport chain within the membrane. Superoxide dismutase suppressed TMT accumulation to a lesser extent than DCP accumulation. The data are interpreted as evidencing the production of intramembrane superoxide in thylakoids.

Control of radical pair lifetimes by microwave irradiation. Application to photosynthetic reaction centres

Abstract Radicals produced by illumination or ionizing radiation are often produced in pairs, which quickly decay by recombination or by diffusion and subsequent reactions. For maximizing the yield of products, and for facilitating the study of reaction pathways, it is desirable to minimize the probability of radical pair recombination. We present a way of controlling the radical pair lifetime through the application of a pulse of resonant microwaves in the presence of a magnetic field. Herewith, two radical pair triplet states are coherently populated, from which the pair cannot recombine directly to the singlet ground state because of spin conservation. We illustrate the method with a photosynthetic photochemical reaction, where we have achieved an increase in the radical pair lifetime of up to two orders of magnitude.

Electron paramagnetic resonance of the primary radical pair [D·+φA·−] in reaction centers of photosynthetic bacteria

Abstract Using continuous wave EPR spectroscopy with a high time resolution, a new short-lived signal at g = 2 is observed in bacterial photosynthetic reaction centers in which electron transfer past the bacteriopheophytin φ A , is blocked. This signal decays with a time constant equal to the rise time of the triplet state of the primary donor 3 D, and is therefore attributed to the primary radical pair [D ·+ φ A ·− ]. Applying the theory of spin-correlated radical pairs, the spectrum could be satisfactorily simulated, yielding the following magnetic interaction parameters between D ·+ and φ A ·− : exchange interaction J D φ = − 0.9 ± 0.1 mT and J D φ = −1.7 ± 0.1 mT for reaction centers of Rhodobacter sphaeroides R26 and Rhodopseudomottas viridis , respectively, and an axial dipolar interaction D D φ = − 3.0 ± 0.5 mT for Rhodopseudomonas viridis . The implications of the magnitude and sign of the exchange parameters for the energetics of photosynthetic electron transfer are discussed.

Combining electrophoresis with detection under ultraviolet light and multiple ultrafiltration for isolation of humic fluorescence fractions.

Polyacrylamide gel electrophoresis of chernozem soil humic acids (HAs) followed by observation under UV (312 nm) excitation light reveals new low molecular weight (MW) fluorescent fractions. Ultrafiltration of HAs sample in 7 M urea on a membrane of low nominal MW retention (NMWR, 5 kDa) was repetitively used for separation of fluorescent and non-fluorescent species. Thirty ultrafiltrates and the final retentate R were obtained. Fluorescence maxima of separate ultrafiltrates were different and non-monotonously changed in the range of 475-505 nm. Fluorescence maxima of less than 490 nm were detected only in the four first utrafiltrates. For further physical-chemical analyses all utrafiltrates were combined into a fraction called UF<5 (NMW<5 kDa). Retentate R demonstrated very weak fluorescence under 270 nm excitation, while fluorescence intensity of UF<5 was about six times higher than of the bulk HAs. Fraction UF<5 was further ultrafiltrated on membranes of MNWR 3 kDa and 1 kDa, yielding three subfractions UF3-5, UF1-3 and UF<1 with NMW 3-5 kDa, 1-3 kDa and <1 kDa, respectively. The validation of the UF procedure was performed by size exclusion chromatography on Sephadex G-25 column. The fluorescence maxima were found to be at 505, 488 and 465 nm for UF3-5, UF1-3 and UF<1, respectively, with increasing of fluorescence intensity from UF3-5 to UF1-3 to UF<1 fraction. EPR analysis showed that the amount of free radicals was the largest in retentate R and drastically decreased in fluorescent ultrafiltrates. The results demonstrate that more than one fluorophore is present in chernozem soil HAs complex.

Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines

Applicability of two lipophilic cyclic hydroxylamines (CHAs), CM-H and TMT-H, and two hydrophilic CHAs, CAT1-H and DCP-H, for detection of superoxide anion radical (O2(∙-)) produced by the thylakoid photosynthetic electron transfer chain (PETC) of higher plants under illumination has been studied. ESR spectrometry was applied for detection of the nitroxide radical originating due to CHAs oxidation by O2(∙-). CHAs and corresponding nitroxide radicals were shown to be involved in side reactions with PETC which could cause miscalculation of O2(∙-) production rate. Lipophilic CM-H was oxidized by PETC components, reducing the oxidized donor of Photosystem I, P700(+), while at the same concentration another lipophilic CHA, TMT-H, did not reduce P700(+). The nitroxide radical was able to accept electrons from components of the photosynthetic chain. Electrostatic interaction of stable cation CAT1-H with the membrane surface was suggested. Water-soluble superoxide dismutase (SOD) was added in order to suppress the reaction of CHA with O2(∙-) outside the membrane. SOD almost completely inhibited light-induced accumulation of DCP(∙), nitroxide radical derivative of hydrophilic DCP-H, in contrast to TMT(∙) accumulation. Based on the results showing that change in the thylakoid lumen pH and volume had minor effect on TMT(∙) accumulation, the reaction of TMT-H with O2(∙-) in the lumen was excluded. Addition of TMT-H to thylakoid suspension in the presence of SOD resulted in the increase in light-induced O2 uptake rate, that argued in favor of TMT-H ability to detect O2(∙-) produced within the membrane core. Thus, hydrophilic DCP-H and lipophilic TMT-H were shown to be usable for detection of O2(∙-) produced outside and within thylakoid membranes.

Free-radical and correlated radical-pair spin-polarized signals in Rhodobacter sphaeroides R-26 reaction centers

Abstract Two g = 2.0 spin-polarized ESR signals with different properties are detected in native and QA-substituted reaction centers from R. sphaeroides R-26. The signal from substituted RCs is attributed to the primary donor cation radical. Native RCs demonstrate a signal with strong temperature dependence which most likely arises from spin-correlated radical pair P+−Q−AFe2+.

Triplet state in photosystem II reaction centers as studied by 130 GHz EPR

Abstract The triplet state in the reaction centers of photosystem II was studied by high-field/high-frequency (130 GHz) EPR in the temperature range 50–90 K. At 50 K, the zero-field splitting parameters of the EPR spectrum correspond well to those of a chlorophyll monomer, in agreement with earlier studies. In the high magnetic field of 4.6 T employed in this study, the g-anisotropy of the triplet state becomes apparent and leads to a shift of the canonical positions of the triplet EPR spectrum. Assuming that triplet g- and zero-field tensors are coaxial, the principal values of the triplet g-tensor are determined to be 2.00324, 2.00306 and 2.00231 with an error of ±0.00004. Lifting this assumption results in higher g-anisotropy. At higher temperatures, the shape of the spectra changes significantly. Triplet excitation hopping involving the accessory chlorophyll BA and PA or PB (equivalents of the special pair bacteriochlorophylls of the bacterial reaction centers) can partially explain those changes, but the most prominent features indicate that also the electron acceptor IA (a pheophytin molecule) must be involved.

Formation of charge separated state P+Q− A and triplet state 3P at low temperature in Rhodobacter sphaeroides (R‐26) reaction centers in which bacteriopheophytin a is replaced by plant pheophytin a

Low temperature optical and photochemical properties of Rhodobacter sphaeroides (R‐26) reaction centers, in which bacteriopheophytin a has been replaced by plant pheophytin a, are reported. Modified reaction centers preserve the ability for photoinduced electron transfer from the primary electron donor P to the primary quinone acceptor QA at 80K. The triplet state ESR signal of modified reaction centers with prereduced QA at 10K shows an electron spin polarization pattern and ZFS parameters analogous to those for the triplet state 3P in non‐treated reaction centers. It was found that at low temperature both P+Q− A and 3P states are formed via a precursor radical pair P+I− in which I is the introduced plant pheophytin molecule. This shows that acceptor systems of bacterial and plant (photosystem II) reaction centers are mutually replacable in structural and functional aspects.

Orientation of the Qy optical transition moment of bacteriopheophytin in Rhodobacter sphaeroides reaction centers

Abstract Time-resolved cw EPR measurements of the Rhodobacter ( Rb ) sphaeroides R-26 reaction center primary donor triplet state excited with plane-polarised light are reported. The pigment composition of the reaction center was chemically modified, so that the bacteriopheophytin molecule in the cofactor branch which is inactive towards electron transfer was replaced by plant pheophytin a . This enabled selective excitation of the bacteriopheophytin and pheophytin molecules, and provided conditions for a high-quality magnetophotoselection study. For the first time, orientation of the Q y optical transition dipole moment relative to the molecular frame of the bacteriopheophytin in the active cofactor branch is determined. Of the four orientations allowed by magnetophotoselection, one was chosen as the most plausible. The corresponding Q y vector is tilted from the bacteriopheophytin tetrapyrrole plane by 15°, and projects onto this plane almost on the y -molecular axis. It is suggested that the deviation of the vector from the molecular plane results from an interaction of bacteriopheophytin with the neighbouring molecule of accessory bacteriochlorophyll.

Involvement of the chloroplast plastoquinone pool in the Mehler reaction

Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler reaction, has been studied using isolated pea thylakoids. The role of the plastoquinone pool in the Mehler reaction was investigated in the presence of dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT), the inhibitor of plastohydroquinone oxidation by cytochrome b6/f complex. Oxygen reduction rate in the presence of DNP-INT was higher than in the absence of the inhibitor in low light at pH 6.5 and 7.6, showing that the capacity of the plastoquinone pool to reduce molecular oxygen in this case exceeded that of the entire electron transfer chain. In the presence of DNP-INT, appearance of superoxide anion radicals outside thylakoid membrane represented approximately 60% of the total superoxide anion radicals produced. The remaining 40% of the produced superoxide anion radicals was suggested to be trapped by plastohydroquinone molecules within thylakoid membrane, leading to the formation of hydrogen peroxide (H2 O2 ). To validate the reaction of superoxide anion radical with plastohydroquinone, xanthine/xanthine oxidase system was integrated with thylakoid membrane in order to generate superoxide anion radical in close vicinity of plastohydroquinone. Addition of xanthine/xanthine oxidase to the thylakoid suspension resulted in a decrease in the reduction level of the plastoquinone pool in the light. The obtained data provide additional clarification of the aspects that the plastoquinone pool is involved in both reduction of oxygen to superoxide anion radicals and reduction of superoxide anion radicals to H2 O2 . Significance of the plastoquinone pool involvement in the Mehler reaction for the acclimation of plants to light conditions is discussed.