I.I. Ivanov

A relay model of lipid peroxidation in biological membranes

It is believed that in lipid peroxidation in membranes an important role is played by unoxygenated hydrocarbon radicals whose movement within the hydrocarbon moiety of the membrane is through intra-and intermolecular relay rearrangements (H-shift). in membranes containing a high concentration of polyunsaturated lipids, mobility may be higher than the lateral diffusion of the lipids allowed by the microviscosity of the membrane. The action mechanism of antioxidants having a side hydrocarbon tail (e.g., tocopherols) rests upon the relay radical reaction, as a multistep process. The initial step is the relay transfer of unoxygenated free radical to the side hydrocarbon chain of antioxidant. In subsequent steps the aroxyl radical is formed by the intramolecular rearrangement of radical in an antioxidant molecule. Hence, the inhibitor interrupts the propagation chain in the membrane by the scavenge of unoxygenated hydrocarbon radical.

Rapid Degradation of the Tetrameric Mn Cluster in Illuminated, PsbO-Depleted Photosystem II Preparations

A “decoupling effect” (light-induced electron transport without O2 evolution) was observed in Ca-depleted photosystem II (PSII(-Ca)) membranes, which lack PsbP and PsbQ (Semin et al. (2008) Photosynth. Res., 98, 235–249). Here PsbO-depleted PSII (PSII(-PsbO)) membranes (which also lack PsbP and PsbQ) were used to examine effects of PsbO on the decoupling. PSII(-PsbO) membranes do not reduce the acceptor 2,6-dichlorophenolindophenol (DCIP), in contrast to PSII(-Ca) membranes. To understand why DCIP reduction is lost, we studied light effects on the Mn content of PSII(-PsbO) samples and found that when they are first illuminated, Mn cations are rapidly released from the Mn cluster. Addition of an electron acceptor to PSII(-PsbO) samples accelerates the process. No effect of light was found on the Mn cluster in PSII(-Ca) membranes. Our results demonstrate that: (a) the oxidant, which directly oxidizes an as yet undefined substrate in PSII(-Ca) membranes, is the Mn cluster (not the YZ radical or P680+); (b) light causes rapid release of Mn cations from the Mn cluster in PSII(-PsbO) membranes, and the mechanism is discussed; and (c) rapid degradation of the Mn cluster under illumination is significant for understanding the lack of functional activity in some PSII(-PsbO) samples reported by others.

Mössbauer spectroscopy of iron metabolism and iron intracellular distribution in liver of rats

Mössbauer spectroscopy was used to investigate the distribution of iron in rat organs and its localisation in liver subcellular fraction. A 57Fe-sucrose complex solution was injected by 0.5 ml doses into tail veins of animals every day, during a 6-day period. Mössbauer spectra were measured in spleen, blood, liver and liver subcellular fractions. The mössbauer spectrum of a spleen sample has two symmetrical doublets, one with delta = 0.6 mm/s and delta = 0.7 mm/s, and the other with delta = 1.0 mm/s and delta = 2.35 mm/s. The Mössbauer spectrum of blood has parameters which are close to those for carboxyhemoglobin and oxyhemoglobin complexes. After the addition of sodium citrate, the proportion of the carboxyhemoglobin complex increases. The Mössbauer spectrum of liver has a two-component pattern with two symmetrical doublets, the first with delta = 0.6 mm/s and delta = 0.63 mm/s and the second with delta = 1.4 mm/s and delta = 3.45 mm/s. The first component, which was identified as ferritin, is present in all subcellular fractions (800 x gav sediment fraction, mitochondrial/lysosomal, microsomal and supernatant fractions), with its greatest content in microsomal fraction. After the addition of NaBH4 to mitochondrial/lysosomal fraction, about 20% of the iron contained in ferritin was reduced. In the Mössbauer spectrum this is reflected by an appearance of a doublet with delta = 0.85 mm/s and delta = 3.7 mm/s.

High-specific binding of Fe(II) at the Mn-binding site in Mn-depleted PSII membranes from spinach

The interaction of Fe(II) and Fe(III) with the ‘high‐affinity Mn‐binding site’ in Mn‐depleted photosystem II (PSII) was investigated using diphenilcarbazide (DPC)/2, 6‐dichlorophenol‐indophenol (DCIP) inhibition assay. Fe(III) was ineffective in the inhibition of DPC‐DCIP reaction while Fe(II) decreased the rate of DCIP photoreduction supported by DPC in the same concentration range as Mn(II). The effectivity of the interaction of Fe(II) with the high affinity Mn‐binding site depends on different anions in the same manner as for Mn(II) and coincides with hierarchy observed for the stimulation of O2 evolution. The Fe(II) binding is accompanied by its oxidation. By using reductants it was shown that the high affinity site contains a redox‐active component and the reduction of this component totally prevents the binding of Fe(II).

pH dependence of the efficiency of binding of iron cations to the donor side of photosystem II

Light-induced interaction of Fe(II) cations with the donor side of Mn-depleted photosystem II (PS II(–Mn)) results in the binding of iron cations and blocking of the high-affinity (HAZ) Mn-binding site. The pH dependence of the blocking was measured using the diphenylcarbazide/2,6-dichlorophenolindophenol test. The curve of the pH dependence is bell-shaped with pK1 = 5.8 and pK2 = 8.0. The pH dependence of the O2-evolution mediated by PS II membranes is also bellshaped (pK2 = 7.6). The pH dependence of the process of electron donation from exogenous donors in PS II(–Mn) was studied to determine the location of the alkaline pH sensitive site of the electron transport chain. The data of the study showed that the decrease in the iron cation binding efficiency at pH > 7.0 during blocking was determined by the donor side of the PS II(–Mn). Mössbauer spectroscopy revealed that incubation of PS II(–Mn) membranes in a buffer solution containing 57Fe(II) + 57Fe(III) was accompanied by binding only Fe(III) cations. The pH dependence of the nonspecific Fe(III) cation binding is also described by the same bell-shaped curve with pK2 = 8.1. The treatment of the PS II(–Mn) membranes with the histidine modifier diethylpyrocarbonate resulted in an increase in the iron binding strength at alkaline pH. It is suggested that blocking efficiency at alkaline pH is determined by competition between OH– and histidine ligand for Fe(III). Because the high-affinity Mn-binding site contains no histidine residue, this fact can be regarded as evidence that histidine is located at another (other than high-affinity) Fe(III) binding site. In other words, this means that the blockage of the high-affinity Mn-binding site is determined by at least two iron cations. We assume that inactivation of oxygen-evolving complex and inhibition of photoactivation in the alkaline pH region are also determined by competition between OH– and a histidine residue involved in coordination of manganese cation outside the high-affinity site.

Comparative study of thermal degradation of iron-sulfur proteins in spinach chloroplasts and membranes of thermophilic cyanobacteria: mössbauer spectroscopy

Mössbauer spectra of chloroplasts isolated from spinach plants grown in a mineral medium enriched with 57Fe and Mössbauer spectra of native membranes of the thermophilic cyanobacterium Synechococcus elongatus contain a broad asymmetric doublet typical of the iron–sulfur proteins of Photosystem (PS) I. Exposure of chloroplasts to temperatures of 20-70°C significantly modifies the central part of the spectra. This spectral change is evidence of decreased magnitude of the quadrupole splitting. However, the thermally induced doublet (ΔQ = 3.10 mm/sec and δ = 1.28 mm/sec) typical of hydrated forms of reduced (divalent) inorganic iron is not observed in spinach chloroplasts. This doublet is usually associated with degradation of active centers of ferredoxin, a surface-exposed protein of PS I. The Mössbauer spectra of photosynthetic membranes of spinach chloroplasts and cyanobacteria were compared using the probability distribution function of quadrupole shift (1/2 quadrupole splitting ΔQ) of trivalent iron. The results of calculation of these functions for the two preparations showed that upon increasing the heating temperature there was a decrease in the probability of the presence of native iron–sulfur centers FX, FA, and FB (quadrupole shift range, 0.43-0.67 mm/sec) in heated preparations. This process was also accompanied by an increase in the probability of appearance of clusters of trivalent iron. This increase was found to be either gradual and continuous or abrupt and discrete in photosynthetic membranes of cyanobacteria or spinach chloroplasts, respectively. The probability of the presence of the iron–sulfur centers FX, FA, and FB in chloroplasts abruptly decreases to virtually to zero within the temperature range critical for inhibition of electron transport through PS I to oxygen. In cyanobacteria, both thermal destruction of iron–sulfur centers of PS I and functional degradation of PS I are shifted toward a higher temperature. The results of this study suggest that the same mechanism of thermal destruction of the PS I core occurs in both thermophilic and mesophilic organisms: destruction of iron–sulfur centers FX, FA, and FB, release of oxidized (trivalent) iron, and its accumulation in membrane-bound iron-oxo clusters.

Inorganic Fe2+ formation upon Fe-S protein thermodestruction in the membranes of thermophilic cyanobacteria: Mössbauer spectroscopy study

A model description of the Mössbauer spectrum (80 K) of native membranes of the thermophilic cyanobacterium Synechococcus elongatus is suggested on the basis of the known values of quadrupole splitting (ΔE Q) and isomer shift (δ Fe) for the iron‐containing components of the photosynthetic apparatus. Using this approach, we found that heating the membranes at 70–80 K results in a decrease of doublet amplitudes belonging to FX, FA, FB and ferredoxin and simultaneous formation of a new doublet with ΔE Q=3.10 mm/s and δ Fe=1.28 mm/s, typical of inorganic hydrated forms of Fe2+. The inhibition of electron transfer via photosystem I to oxygen, catalyzed by ferredoxin, occurs within the same range of temperatures. The data demonstrate that the processes of thermoinduced Fe2+ formation and distortions in the photosystem I electron transport in the membranes are interrelated and caused mainly by the degradation of ferredoxin. The possible role of Fe2+ formation in the damage of the photosynthetic apparatus resulting from heating and the action of other extreme factors is discussed.

Effect of calcium chelators on the formation and oxidation of the slowly relaxing reduced plastoquinone pool in calcium-depleted PSII membranes. Investigation of the F0 yield

The F0 fluorescence yield in intact photosystem II (PSII), Ca-depleted PSII (PSII(-Ca/NaCl)), and Mn-depleted PSII membranes was measured before and after dim light treatment (1–2 min), using flash-probe fluorescence and fluorescence induction kinetic measurements. The value of F0 after the light treatment (F’0) was larger than F0 in dark-adapted PSII membranes and depended on the appearance of the slowly relaxing, reduced plastoquinone pool (t1/2 = 4 min) formed during preillumination, which was not totally reoxidized before the F’0 measurement. In PSII(-Ca/NaCl) such a pool also appeared, but the F’0 yield was even higher than in intact PSII membranes. In Mn-depleted PSII membranes, the pool did not form. Interestingly, the yield of F’0 in Ca-depleted PSII membranes prepared using chelators (EGTA and citrate) or containing 5 mM EGTA was significantly lower than in PSII(-Ca/NaCl) samples prepared without chelators. These data indicate that chelators inhibit the reduction of QA and QB and formation of the slowly relaxing plastoquinone pool, or alternatively they increase the rate of its oxidation. Such an effect can be explained by coordination of the chelator molecule to the Mn cluster in PSII(-Ca/NaCl) membranes, rather than different amounts of residual Ca2+ in the membranes (with or without the chelator), since the remaining oxygen-evolving activity (∼15%) in PSII(-Ca/NaCl) samples did not depend on the presence of the chelator. Thus, chelators of calcium cations not only have an effect on the EPR properties of the S2 state in PSII(-Ca/NaCl) samples, but can also influence the PSII properties determining the rate of plastoquinone pool reduction and/or oxidation. The effect of some toxic metal cations (Cd, Cu, Hg) on the formation of the slowly relaxing pool in PSII membranes was also studied.

Comparative Study of Effects of Artificial Electron Donors on the AT-Band of Photosystem II Thermoluminescence

Extraction of the Mn-cluster from photosystem II (PS II) inhibits the main bands of thermoluminescence and induces a new AT-band at –20°C. This band is attributed to the charge recombination between acceptor QA− and a redoxactive histidine residue on the donor side of PS II. The effect of Mn(II) and Fe(II) cations as well as the artificial donors diphenylcarbazide and hydroxylamine on the AT-band of thermoluminescence was studied to elucidate the role of the redoxactive His residue in binding to the Mn(II) and Fe(II). At the Mn/PS II reaction center (RC) ratio of 90 : 1 and Fe/PS II RC ratio of 120 : 1, treatment with Mn(II) and Fe(II) causes only 60% inhibition of the AT-band. Preliminary exposure of Mn-depleted PS II preparations to light in the presence of Mn(II) and Fe(II) causes binding of the cations to the high-affinity Mn-binding site, thereby inhibiting oxidation of the His residue involved in the AT -band formation. The efficiency of the AT-band quenching induced by diphenylcarbazide and hydroxylamine is almost an order of magnitude higher than the quenching efficiency of Mn(II) and Fe(II). Our results suggest that the redox-active His is not a ligand of the high-affinity site and does not participate in the electron transport from Mn(II) and Fe(II) to YZ . The concentration dependences of the AT-band inhibition by Mn(II) and Fe(II) coincide with each other, thereby implying specific interaction of Fe(II) with the donor side of PS II.

Characteristic features of the interaction between Fe(II) cations and the, donor side of the manganese-depleted photosystem II

Ferrous iron cations Fe(II) can effectively bind to the donor side of the manganese-depleted photosystem II (PSII(-Mn)) and in this way block electron transfer from diphenylcarbazide (DPC) to the major donor for P680, YZ. The present study was focused on the characteristic features of this process. The oxidation and subsequent binding of Fe(II) cations to PSII(-Mn) may proceed in the absence of an artificial electron acceptor, and therefore we investigated the role of O2 as a putative endogenous acceptor. Oxygen was shown to participate in the blockade of YZ by Fe cations, apparently as a structural element of Fe cluster formed at the donor side of PSII(-Mn). The kinetic study of blocking YZ by Fe(II) as dependent on light intensity demonstrated that the quantum efficiency of Fe cations binding to the donor side of PSII(-Mn) considerably exceeded that of Mn cations. We also compared the possibilities of extracting the native Mn cluster and reconstructed Fe cations from PSII and an alternative electron transport from DPC to P680+ under the conditions of the YZ blockade by Fe cations. Neither an alternative donor for P680, YD , nor cytochrome b559 participated in the latter process. As a whole, our evidence shows that many features of binding Fe cation to the donor side of PSII(-Mn) are in common with photoassembling the Mn cluster.