Search

Summary Using high-throughput screening we identified small molecules that suppress superoxide and/or H 2 O 2 production during reverse electron transport through mitochondrial respiratory complex I (site I Q ) without affecting oxidative phosphorylation (suppressors of site I Q electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site I Q is a normal contributor to mitochondrial superoxide-H 2 O 2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H 2 O 2 production by site I Q is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H 2 O 2 production by site I Q is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site I Q to cell physiology and pathology and have great potential as therapeutic leads.

The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.

Mitochondria constitute an important topic of biomedical enquiry (one paper in every 154 indexed in PubMed since 1998 is retrieved by the keyword 'mitochondria') because of widespread recognition of their importance in cell physiology and pathology. Mitochondrial dysfunction is widely implicated in ageing and in the diseases of ageing, through dysfunction in adenosine triphosphate (ATP) synthesis, Ca(2+) homeostasis, central metabolic pathways or radical production. Nonetheless, the mechanisms and regulation of superoxide and hydrogen peroxide formation by mitochondria remain poorly described. Measurement of the capacities of different sites of superoxide and hydrogen peroxide production in isolated skeletal muscle mitochondria show that the maximum capacities of sites in complexes I, II and III and in several associated redox enzymes greatly exceed the native rates observed in the absence of respiratory chain inhibitors. In vitro, the native rates and the relative importance of different sites both depend on the substrate being oxidized, with sites IQ, IIF, GPDH, IF and IIIQo each being important with particular substrates. The techniques involved in measuring rates from each site should become applicable to cell cultures and in vivo in the future.

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.

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.

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

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 H(2)O(2) 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 II(F)). 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.

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 (C(12)R1) 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 C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 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 C(12)R1 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.

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 β-structures and α-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core.

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.