Irina V. Perevoshchikova

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Background: Complex II is not considered a significant contributor to mitochondrial ROS production. Results: Complex II generates ROS in both the forward reaction, from succinate, and the reverse reaction, from the reduced ubiquinone pool. Conclusion: Occupancy and reduction state of the flavin dictate its ROS producing behavior. Significance: Based on the maximum rates observed, complex II may be a contributor to physiological ROS production. Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H2O2 at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site IIF). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

Suppressors of superoxide production from mitochondrial complex III

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species (ROS), which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies but its role remains controversial. Using high-throughput screening we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

Peak intensity analysis as a method for estimation of fluorescent probe binding to artificial and natural nanoparticles: Tetramethylrhodamine uptake by isolated mitochondria

A modified version of fluorescence correlation spectroscopy (FCS) closely related to the photon counting histogram (PCH) method, which is used in the case of a mixture of molecules with similar diffusion coefficients, was applied here for analyzing the binding of the potential-sensitive dye tetramethylrhodamine ethyl ester, TMRE, to isolated mitochondria both in energized and deenergized states. Fluorescence time traces of suspensions of TMRE-doped mitochondria representing sequences of peaks of different intensity appeared to be similar to those of fluorescent beads and TMRE-doped latex particles. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude thus substantially enhancing the resolution of the method. The statistics of the brightness of identical fluorescent particles reflecting their random walk through the confocal volume was described by a simple analytical equation which enabled us to perform the peak intensity analysis (PIA) of TMRE-doped mitochondria. The validity of PIA was tested with fluorescent beads of different sizes and TMRE-doped latex particles. Mitochondrial energization in the presence of TMRE led to the increase in the number and the intensity of the peaks in fluorescence time traces, the PIA of which allowed us to determine mitochondrial membrane potential and additionally a number of mitochondrial particles per ml of the suspension. The value of the membrane potential on a single mitochondrion was estimated to be about 180 mV in agreement with the data related to mitochondrial suspensions. Importantly, the PIA method required less than 1 microgram of mitochondrial protein per measurement.

Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin.

Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY‐FL‐ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY‐FL‐ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY‐FL‐ATP accumulation, while a 15‐residue peptide corresponding to the N‐terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase‐induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria.

Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension

The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 μM) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 μM safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(τ→0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 μg).

Fluorescence correlation spectroscopy in biology, chemistry, and medicine

This review describes the method of fluorescence correlation spectroscopy (FCS) and its applications. FCS is used for investigating processes associated with changes in the mobility of molecules and complexes and allows researchers to study aggregation of particles, binding of fluorescent molecules with supramolecular complexes, lipid vesicles, etc. The size of objects under study varies from a few angstroms for dye molecules to hundreds of nanometers for nanoparticles. The described applications of FCS comprise various fields from simple chemical systems of solution/micelle to sophisticated regulations on the level of living cells. Both the methodical bases and the theoretical principles of FCS are simple and available. The present review is concentrated preferentially on FCS applications for studies on artificial and natural membranes. At present, in contrast to the related approach of dynamic light scattering, FCS is poorly known in Russia, although it is widely employed in laboratories of other countries. The goal of this review is to promote the development of FCS in Russia so that this technique could occupy the position it deserves in modern Russian science.

Interaction of tetrasubstituted cationic aluminum phthalocyanine with artificial and natural membranes.

A study of the properties of water-soluble tetrasubstituted cationic aluminum phthalocyanine (AlPcN4) revealed efficient binding of this photosensitizer to phospholipid membranes as compared with tetrasulfonated aluminum and zinc phthalocyanine complexes. This also manifested itself in enhanced photodynamic activity of AlPcN4 as measured by the photosensitized damage of gramicidin channels in a planar bilayer lipid membrane. The largest difference in the photodynamic activity of cationic and anionic phthalocyanines was observed in a membrane containing negatively charged lipids, thereby pointing to significant contribution of electrostatic interactions to the binding of photosensitizers to a membrane. Fluoride anions suppressed the photodynamic activity and binding to membrane of both tetraanionic and tetracationic aluminum phthalocyanines, which supports our hypothesis that interaction of charged metallophthalocyanines with phospholipid membranes is mostly determined by coordination of the central metal atom with the phosphate group of lipid.

Effect of liposomes on energy-dependent uptake of the antioxidant SkQR1 by isolated mitochondria

The mitochondria-targeted antioxidant SkQR1 composed of a plastoquinone part covalently bound to a cationic rhodamine 19 moiety via a decane linker was previously shown to effectively protect brain and kidney from ischemia injury accompanying generation of reactive oxygen species. In the present paper the energy-dependent SkQR1 uptake by isolated rat liver mitochondria was studied by fluorescence correlation spectroscopy peak intensity analysis (FCS PIA). This approach can be used to measure the number of fluorescent molecules per single mitochondrion. A large portion of SkQR1 appeared to be taken up by mitochondria in an energy-independent fashion because of its high affinity to membranes. Liposomes were found to compete effectively with mitochondria for the energy-independent SkQR1 binding, thereby facilitating, as an “SkQR1-buffer”, observation of energy-dependent SkQR1 accumulation in mitochondria. The rate of energy-dependent SkQR1 uptake by mitochondria observed in the presence of liposomes was rather low (minutes) which was apparently due to slow redistribution of SkQR1 between liposomal and mitochondrial membranes. This can explain the low rate of staining of mitochondria by SkQR1 in living cells containing, besides mitochondria, other membrane components (endoplasmic reticulum, Golgi membranes, endosomes, lysosomes, etc.) which can compete with mitochondria for the energy-independent SkQR1 binding.

Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes

Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to beta-structures and alpha-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core.