ROS signaling capacity of cytochrome bc1: Opposing effects of adaptive and pathogenic mitochondrial mutations.

Cytochrome bc1, also known as mitochondrial complex III, is considered to be one of the important producers of reactive oxygen species (ROS) in living organisms. Under physiological conditions, a certain level of ROS produced by mitochondrial electron transport chain (ETC) might be beneficial and take part in cellular signaling. However, elevated levels of ROS might exhibit negative effects, resulting in cellular damage. It is well known that inhibiting the electron flow within mitochondrial complex III leads to high production of ROS. However, superoxide production by cytochrome bc1 in a non-inhibited system remained controversial. Here, we propose a novel method for ROS detection in ETC hybrid system in solution comprising bacterial cytochrome bc1 and mitochondrial complex IV. We clearly show that non-inhibited cytochrome bc1 generates ROS and that adaptive and pathogenic mitochondrial mutations suppress and enhance ROS production, respectively. We also noted that cytochrome bc1 produces ROS in a rate-dependent manner and that the mechanism of ROS generation changes according to the rate of operation of the enzyme. This dependency has not yet been reported, but seems to be crucial when discussing ROS signaling originating from mitochondria.

The Ability of Functionalized Fullerenes and Surface-Modified TiO2 Nanoparticles to Photosensitize Peroxidation of Lipids in Selected Model Systems.

Photochemical properties of a new class of inorganic nanoparticles, namely a cationic C60 fullerene substituted with three quaternary pyrrolidinium groups (BB6) and a surface-modified nanocrystalline TiO2 with bromopyrogallol red (Brp@TiO2 ) were examined for their effectiveness in photogenerating singlet oxygen and free radicals. In particular, their ability to photosensitize peroxidation of unsaturated lipids was analyzed in POPC:cholesterol liposomes and B16 mouse melanoma cells employing a range of spectroscopic and analytical methods. Because melanoma cells typically are pigmented, we examined the effect of melanin on the photosensitized peroxidation of lipids in liposomes and B16 melanoma cells, mediated by BB6 and Brp@TiO2 nanoparticles. The obtained results suggest that peroxidation of unsaturated lipids, photosensitized by BB6 occurs mainly, although not exclusively, via Type II mechanism involving singlet oxygen. On the other hand, if surface-modified TiO2 is used as a photosensitizer, Type I mechanism of lipid peroxidation dominates, as indicated by the predominant formation of the free radical-dependent cholesterol oxidation products. The protective effect of melanin was particularly evident when BB6 was used as a photosensitizer, suggesting that melanin could efficiently interfere with Type II processes.

Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems.

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.

A peculiar phase transition of plasmalogen bilayer membrane under high pressure.

The bilayer phase transition of plasmalogen, monounsaturated plasmenylcholine 1-O-1'-(Z)-octadecenyl-2-oleoyl-sn-glycero-3-phosphocholine (Plg-SOPC), was examined by differential scanning calorimetry, high-pressure transmittance, and fluorescence techniques. The bilayer properties of Plg-SOPC such as the temperature-pressure phase diagram, the thermodynamic quantities of the transition, and the location of a fluorescent membrane probe in the bilayer, were compared with those of a similar phospholipid 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). It turned out that a vinyl-ether bond in the sn-1 position of the glycerol backbone in the Plg-SOPC molecule produces a peculiar phase transition under high pressure and significantly affects the membrane properties.

Trifluoperazine causes a disturbance in glycerophospholipid monolayers containing phosphatidylserine (PS): effects of pH, acyl unsaturation, and proportion of PS.

We have studied the interaction of trifluoperazine (TFP) with monolayers of various glycerophospholipids at 37 degrees C. TFP (1-10 microM) had little effect on surface pressure/molecular area isotherms in monolayers (on pure water) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine but greatly increased the mean molecular area (mma) of dipalmitoylphosphatidylserine; the increment was greatest between 0 and 1 microM, and a further increase to 10 microM TFP gave only a slight increase in mma. With phosphatidylserine (PS)-containing stearoyl and varying acyls in the sn-1 and -2 positions, respectively, TFP increased the mma in a manner that depended on the number of double bonds and chain length. In mixtures of DPPC with two of these PS species the TFP-induced mma of the monolayers (on buffer, pH 7.4) increased linearly with the proportion of PS. Both PS and TFP have ionizable groups, and the TFP-induced mma increase had optima at pH 5.0 and 7.0. We conclude that the TFP-PS interaction is mainly, but not entirely, driven by electrostatic interactions between the TFP cation and PS headgroup anion, with an insertion of the phenothiazine moiety among the acyls in the monolayer that depends on the packing of the acyls.

Spectroscopic properties and reactivity of free radical forms of A2E.

A pyridinium bisretinoid (A2E) is the only identified blue-absorbing chromophore of retinal lipofuscin that has been linked to its aerobic photoreactivity and phototoxicity. Pulse radiolysis has been used to study both the one-electron oxidation and the one-electron reduction of A2E in aqueous micellar solutions. The reduction to the semireduced A2E (lambda(max) broad and between 500 and 540 nm) was achieved with formate radicals and the subsequent decay of A2E* was slow (over hundreds of milliseconds) via complex kinetics. The long lifetime of the A2E* should facilitate its reactions with other biomolecules. For example, with oxygen, the A2E* produced the superoxide radical anion with a rate constant of 3 x 10(8) M(-1) s(-1). The A2E was also reduced by the NAD radical, the corresponding rate constant being 2.3 x 10(8) M(-1) s(-1). Other experiments showed that the one-electron reduction potential of A2E lies in the range -640 to -940 mV. The semioxidized form of A2E (lambda(max) 590 nm) was formed via oxidation with the Br2*- radical and had a much shorter lifetime than the semireduced form. With strongly oxidizing peroxyl radicals (CCl3O2*) our kinetic data suggest the formation of a radical adduct followed by dissociation to the semioxidized A2E. With milder oxidizing peroxyl radicals such as that from methanol, our results were inconclusive. In benzene we observed an efficient oxidation of zeaxanthin to its radical cation by the A2E radical cation; this may be relevant to a detrimental effect of A2E in vision.