BBA - Bioenergetics (v.1797, #11)
Editorial Board (i).
Mitochondrial Ca2+ transport and permeability transition in zebrafish (Danio rerio) by Luca Azzolin; Emy Basso; Francesco Argenton; Paolo Bernardi (1775-1779).
We have studied mitochondrial Ca2+ transport and the permeability transition (PT) in the teleost zebrafish (Danio rerio), a key model system for human diseases. Permeabilized zebrafish embryo cells displayed a mitochondrial energy-dependent Ca2+ uptake system that, like the Ca2+ uniporter of mammals, was inhibited by ruthenium red. Zebrafish mitochondria underwent a Ca2+-dependent PT that displayed Pi-dependent desensitization by cyclosporin A, and responded appropriately to key modulators of the mammalian PT pore (voltage, pH, ubiquinone 0, dithiol oxidants and cross linkers, ligands of the adenine nucleotide translocator, arachidonic acid). Opening of the pore was documented in intact cells, where it led to death that could largely be prevented by cyclosporin A. Our results represent a necessary step toward the use of zebrafish for the screening and validation of PTP inhibitors of potential use in human diseases, as recently shown for collagen VI muscular dystrophy [Telfer et al., 2010].
Keywords: Mitochondria; Permeability Transition; Cyclosporin A; Disease models; Drug screening;
Excitation transfer connectivity in different purple bacteria: A theoretical and experimental study by Matthieu de Rivoyre; Nicolas Ginet; Pierre Bouyer; Jérôme Lavergne (1780-1794).
Photosynthetic membranes accommodate densely packed light-harvesting complexes which absorb light and convey excitation to the reaction center (RC). The relationship between the fluorescence yield (φ) and the fraction (x) of closed RCs is informative about the probability for an excitation reaching a closed RC to be redirected to another RC. In this work, we have examined in this respect membranes from various bacteria and searched for a correlation with the arrangement of the light-harvesting complexes as known from atomic force or electron microscopies. A first part of the paper is devoted to a theoretical study analyzing the φ(x) relationship in various models: monomeric or dimeric RC–LH1 core complexes, with or without the peripheral LH2 complexes. We show that the simple “homogeneous” kinetic treatment used here agrees well with more detailed master equation calculations. We also discuss the agreement between information derived from the present technique and from singlet annihilation experiments. The experimental results show that the enhancement of the cross section of open RCs due to excitation transfer from closed units varies from 1.5 to 3 depending on species. The ratio of the core to core transfer rate (including the indirect pathway via LH2) to the rate of trapping in open units is in the range of 0.5 to 4. It is about 1 in Rhodobacter sphaeroides and does not increase significantly in mutants lacking LH2—despite the more numerous contacts between the dimeric core complexes expected in this case. The connectivity in this bacterium is due in good part to the fast transfer between the two partners of the dimeric (RC–LH1–PufX)2 complex. The connectivity is however increased in the carotenoidless and LH2-less strain R26, which we ascribe to an anomalous LH1. A relatively high connectivity was found in Rhodospirillum photometricum, although not as high as predicted in the calculations of Fassioli et al. (2010). This illustrates a more general discrepancy between the measured efficiency of core to core excitation transfer and theoretical estimates. We argue that the limited core to core connectivity found in purple bacteria may reflect a trade-off between light-harvesting efficiency and the hindrance to quinone diffusion that would result from too tightly packed LH complexes.Display Omitted►Cross-section enhancement brought about by antenna connectivity ranges from 1.5 to 3 ►In Rhodobacter sphaeroides (closed RCs) the excitation visits ~ two dimeric complexes ►Effective transfer between core complexes is markedly slower than generally believed ►Chemical-kinetics-like and more refined modeling of excitation migration are in good agreement
Keywords: Bacteriochlorophyll fluorescence; Excitation transfer; Light harvesting; LH1; LH2; Photosynthetic bacteria; Supramolecular organization;
BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: Role of permeability transition and SH-redox regulation by Tatiana Brustovetsky; Tsyregma Li; Youyun Yang; Jiang-Ting Zhang; Bruno Antonsson; Nickolay Brustovetsky (1795-1806).
BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/ oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state.The graphical abstract shows a scheme, illustrating our major findings. We consider a simplified model in which BAX oligomers form proteinaceous pores in the OMM. The minimal number of BAX molecules required for BAX pore formation is unknown. Therefore, in our model the number of BAX molecules in the BAX oligomer is arbitrary. Our experiments revealed that BAX can self-integrate and self-oligomerize in the OMM, producing only limited OMM permeabilization. DTT prevents formation of disulfide bridges between BAX molecules and thus inhibits BAX insertion/oligomerization. Ca2+ and tBID increase amount of inserted/oligomerized BAX. Ca2+ induces the mPT that causes mitochondrial swelling and probably stretching of the OMM. This might lead to dilation of the pore formed by BAX oligomer similar to stretch-activated channels (Sachs, 2010). On the other hand, tBID might interact with BAX molecules composing BAX oligomer, change conformation of the oligomer, and cause pore dilation. It is conceivable that tBID stimulates formation of larger BAX pores permeable for both Cyt c and Smac/DIABLO, while Ca2+ stimulates formation of smaller BAX pores preferentially permeable for Cyt c. With tBID-stimulated BAX pore formation, DTT could attenuate OMM permeability by inhibiting BAX insertion/oligomerization and decreasing the size of the BAX pores. In both cases, with Ca2+ in this study and with tBID in our previous paper (Brustovetsky et al., 2003), Bcl-xL strongly inhibited Cyt c release but failed to inhibit BAX insertion/oligomerization. This suggests that Bcl-xL blocks the pore formed by BAX oligomer in the OMM. Alternatively, in the case of tBID, Bcl-xL might compete for tBID with BAX oligomer, leading to tBID dissociation from BAX and constriction of the pore. Correspondingly, a decrease in the size of the BAX pore might play a decisive role in limiting OMM permeability. Overall, our results strongly suggest that BAX-mediated OMM permeabilization in brain mitochondria can be modulated by the mPT and by SH-redox state. Subsequently, induction of the mPT, increased ROS generation and oxidation of critical SH-groups could significantly augment BAX-mediated permeabilization of the OMM and thus promote neuronal apoptosis in various neurodegenerative diseases, stroke, and traumatic brain injury.Display Omitted▶ BAX self-integrates/oligomerizes in the OMM with miniscule cytochrome c release. ▶ Ca2+ stimulates BAX insertion and cytochrome c release. ▶ Ca2+ effect depends on induction of the mitochondrial permeability transition. ▶ Bcl-xL does not prevent BAX insertion but attenuates cytochrome c release. ▶ DTT inhibits BAX insertion and the release of cytochrome c and Smac/DIABLO.
Keywords: Mitochondria; Calcium; BAX; BID; Bcl-xL; Permeability transition;
Singlet oxygen scavenging activity of plastoquinol in photosystem II of higher plants: Electron paramagnetic resonance spin-trapping study by Deepak Kumar Yadav; Jerzy Kruk; Rakesh Kumar Sinha; Pavel Pospíšil (1807-1811).
Singlet oxygen (1O2) scavenging activity of plastoquinol in photosystem II (PSII) of higher plants was studied by electron paramagnetic resonance (EPR) spin-trapping technique. It is demonstrated here that illumination of spinach PSII membranes deprived of intrinsic plastoquinone results in 1O2 formation, as monitored by TEMPONE EPR signal. Interestingly, the addition of exogenous plastoquinol (PQH2-1) to PQ-depleted PSII membranes significantly suppressed TEMPONE EPR signal. The presence of exogenous plastoquinols with a different side-chain length (PQH2-n, n isoprenoid units in the side chain) caused a similar extent of 1O2 scavenging activity. These observations reveal that plastoquinol exogenously added to PQ-depleted PSII membranes serves as efficient scavenger of 1O2.►Illumination of PQ-depleted PSII membranes results in 1O2 formation. ►The addition of exogenous plastoquinol significantly suppressed 1O2 formation. ►Plastoquinol acts as efficient 1O2 scavenger in PSII under oxidative stress.
Keywords: Electron paramagnetic resonance; Photoinhibition; Photosystem II; Plastoquinol; Singlet oxygen; Spin trap;
Dimerisation of the Rhodobacter sphaeroides RC–LH1 photosynthetic complex is not facilitated by a GxxxG motif in the PufX polypeptide by Lucy I. Crouch; Katherine Holden-Dye; Michael R. Jones (1812-1819).
In purple photosynthetic bacteria the initial steps of light energy transduction take place in an RC–LH1 complex formed by the photochemical reaction centre (RC) and the LH1 light harvesting pigment-protein. In Rhodobacter sphaeroides, the RC–LH1 complex assembles in a dimeric form in which two RCs are surrounded by an S-shaped LH1 antenna. There is currently debate over the detailed architecture of this dimeric RC–LH1 complex, with particular emphasis on the location and precise function of a minor polypeptide component termed PufX. It has been hypothesised that the membrane-spanning helical region of PufX contains a GxxxG dimerisation motif that facilitates the formation of a dimer of PufX at the interface of the RC–LH1 dimer, and more specifically that the formation of this PufX dimer seeds assembly of the remaining RC–LH1 dimer (J. Busselez et al., 2007). In the present work this hypothesis was tested by site directed mutagenesis of the glycine residues proposed to form the GxxxG motif. Mutation of these glycines to leucine did not decrease the propensity of the RC–LH1 complex to assemble in a dimeric form, as would be expected from experimental studies of the effect of mutation on GxxxG motifs in other membrane proteins. Indeed increased yields of dimer were seen in two of the glycine-to-leucine mutants constructed. It is concluded that the PufX from Rhodobacter sphaeroides does not contain a genuine GxxxG helix dimerisation motif.Display Omitted►The PufX protein is a key component of the Rhodobacter photosystem. ►PufX strongly affects structural and functional organisation. ►Dimerisation of the RC-LH1 complex is not mediated by a GxxxG sequence motif. ►The GxxxGxxxG sequence does not constitute a genuine GxxxG dimerisation motif.
Keywords: Photosynthesis; Rhodobacter sphaeroides; Reaction centre; Light harvesting; PufX; GxxxG;
Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc 1 by Marcin Sarewicz; Arkadiusz Borek; Ewelina Cieluch; Monika Świerczek; Artur Osyczka (1820-1827).
In addition to its bioenergetic function of building up proton motive force, cytochrome bc 1 can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQo) formed at the quinone oxidation/reduction Qo site (Qo) as a result of single-electron oxidation of quinol by the iron–sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b L (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc 1 that severely impeded the oxidation of FeS by cytochrome c 1, changed density of FeS around Qo by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Qo and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQo that present a risk of generation of superoxide by cytochrome bc 1. Isolation of this reaction sequence from multiplicity of possible reactions at Qo helps to better understand conditions under which complex III might contribute to ROS generation in vivo.
Keywords: Cytochrome; Mitochondria; ROS; Electron transfer; Quinone; Kinetic modeling;
Proton transport coupled ATP synthesis by the purified yeast H+-ATP synthase in proteoliposomes by Kathrin Förster; Paola Turina; Friedel Drepper; Wolfgang Haehnel; Susanne Fischer; Peter Gräber; Jan Petersen (1828-1837).
The H+/ATP synthase from yeast mitochondria, MF0F1, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF0F1 liposomes were energized by acid–base transitions (ΔpH) and a K+/valinomycin diffusion potential (Δφ). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120 s− 1 at a transmembrane pH difference of 3.2 units (at pHin = 4.8 and pHout = 8.0) and a Δφ of 133 mV (Nernst potential). Functional studies showed that the monomeric MF0F1 was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of KM on pHout indicated that the substrate for ATP synthesis is the monoanionic phosphate species H2PO4 −.►Purified mitochondrial H+-ATPsynthase from yeast catalyzes rates up to 120 s− 1. ►The monomer of MF0F1 is fully active in ATP synthesis. ►The phosphate species in ATP synthesis is H2PO4 −. ►The KM of H2PO4 − in ATP synthesis is 120 μM.
Keywords: ATP synthesis; Mitochondrial H+-ATP synthase; KM of phosphate; MF0F1; Reconstitution; Saccharomyces cerevisiae;
Erratum to “Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study” [Biochim. Biophys. Acta 1797 (2010)1617–1626] by N.S. Ponomarenko; O.G. Poluektov; E.J. Bylina; J.R. Norris (1838-1840).