Irina B. Klenina

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

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.

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+.

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.

Mechanism of photoinduced electron transfer in photosystem II reaction centers

The shape of the EPR spectrum of the triplet state of photosystem II reaction centers with a singly reduced primary acceptor complex QAFe2+ was studied. It was shown that the spectroscopic properties do not significantly change when the relaxation of the primary acceptor is accelerated and when the magnetic interaction between the reduced quinone molecule QA and the nonheme iron ion Fe2+ is disrupted. This observation confirmed the earlier conclusion that the anisotropy of the quantum yield of the triplet state is the main cause of the anomalous shape of the EPR spectrum. A scheme of primary processes in photosystem II that is consistent with the observed properties of the EPR spectrum of the triplet state is discussed.

Photoselection in ESP Spectra of Photosynthetic Reaction Centers

The first relatively stable stage of photoinduced electron transfer in reaction centers (RCs) of many photosynthetic bacteria is P+Q A - , where P is the so — called primary donor (dimer of bacteriochlorophyll molecules), and Q A - primary acceptor, a molecule of quinone. As early as 1977 [1], it was reported that when the normally present magnetic interaction between Q A - and paramagnetic Fe2+ — ion is disrupted, an electron spin — polarised (ESP) photoinduced signal may be detected using time — resolved EPR. This signal was subsequently attributed to the state P+Q A - , which since 1987 [2] is considered as a spin — correlated radical pair (SCRP). Simulation of experimental ESP spectra of P+Q A - using the SCRP model, will provide information on the structural organisation of RC and on interactions between its cofactors. Recently we observed that the shape of the ESP signal of P+Q A - in Zn2+ — substituted RCs of Rb. sphaeroides R26 strongly depends on the wavelength of the excitation laser flash. This dependence arises due to the effect of photoselection, i.e., selective excitation of certain orientations of RCs in the sample by plane — polarised laser light. When taking photoselection into account, the SCRP model satisfactorily describes the quite different ESP signals for light polarised parallel and perpendicular to the magnetic field of the spectrometer.

Photoselection Effects in Epr-Detected Triplet States of Photosynthetic Pigment Molecules

Photosynthetic reaction centers (RCs) transform the energy of light absorbed by pigment molecules into chemical energy. RCs of bacteria are best characterised both functionally and structurally. In them, excitation of the primary donor P (dimer of bacteriochlorophylls) leads to a sequence of electron-transfer reactions to intermediate acceptor (bacteriopheophytin) and then to a quinone primary acceptor. When electron transfer past the intermediate acceptor is blocked, reverse electron transfer to oxidised P occurs, populating the triplet state of P (at 1<77 K with almost-unity yield). Though the triplet states are the side products, their properties make them excellent probes of the RC photochemistry (for review see [1, 2]). Magnetophotoselection, MPS, or EPR detection of triplet states excited with polarised light, proved to be a powerful technique for the studies of the triplet states. Usually MPS is performed utilising cw EPR detection [1]. First application of time-resolved direct-detection EPR (DD-EPR) to the study of a synthetic aromatic molecule was reported in [3]. DD-EPR is advantageous in such studies, for it is essentially free from a drawback of cw EPR MPS, the latter’s strong dependence on spin-lattice relaxation. This enables investigations in a wide temperature range. Here we report on the first observation of DD-EPR MPS of the primary donor triplet state of Rb. sphaeroides R26 RCs and D1/D2-cyt b-559 complexes of plant photosystem II.