BBA - Bioenergetics (v.1807, #12)

Editorial by Peter R. Rich (1505-1506).

50 years ago Peter Mitchell proposed the chemiosmotic hypothesis for which he was awarded the Nobel Prize for Chemistry in 1978. His comprehensive review on chemiosmotic coupling known as the first “Grey Book”, has been reprinted here with permission, to offer an electronic record and easy access to this important contribution to the biochemical literature. This remarkable account of Peter Mitchell's ideas originally published in 1966 is a landmark and must-read publication for any scientist in the field of bioenergetics. As far as was possible, the wording and format of the original publication have been retained. Some changes were required for consistency with BBA formats though these do not affect scientific meaning. A scanned version of the original publication is also provided as a downloadable file in . See also Editorial in this issue by Peter R. Rich. Original title: CHEMIOSMOTIC COUPLING IN OXIDATIVE AND PHOTOSYNTHETIC PHOSPHORYLATION, by Peter Mitchell, Glynn Research Laboratories, Bodmin, Cornwall, England.
Keywords: Chemiosmotic theory; Proton pumping; Mitochondria; Respiration; Photosynthesis; Oxidative phosphorylation; Peter Mitchell;

In this work we address the question whether light-induced changes in the Mg(II) content in the chloroplast lumen can modulate the electron donation to photosystem I, in particular the electrostatic interaction between plastocyanin (Pc) and the photosystem 1 subunit PsaF. For this, we have used 2D NMR spectroscopy to study the binding of Mg(II) ions and the isolated luminal domain of PsaF to 15  N-labelled Pc. From the chemical-shift perturbations in the 1H-15  N HSQC spectra, dissociation constants of (4.9 ± 1.7) mM and (1.4 ± 0.2) mM were determined for the Pc-Mg(II) and Pc-PsaF complexes, respectively. In both cases, significant chemical-shift changes were observed for Pc backbone amide groups belonging to the two acidic patches, residues 42–45 and 59–61. In addition, competitive effects were observed upon the addition of Mg(II) ions to the Pc-PsaF complex, further strengthening that Mg(II) and PsaF bind to the same region on Pc. To structurally elucidate the Mg(II) binding site we have utilized Mn(II) as a paramagnetic analogue of Mg(II). The paramagnetic relaxation enhancement induced by Mn(II) results in line broadening in the Pc HSQC spectra which can be used to estimate distances between the bound ion and the affected nuclear spins. The calculations suggest a location of the bound Mn(II) ion close to Glu43 in the lower acidic patch, and most likely in the form of a hexaquo complex embedded within the hydration shell of Pc. The results presented here suggest a specific binding site for Mg(II) that may regulate the binding of Pc to photosystem 1 in vivo.Display Omitted► Dissociation constants for the binding of Mg(II) and the luminal domain of PsaF to plastocyanin. ► Competitive binding between Mg(II) and PsaF to the acidic patches of plastocyanin. ► Exploration of Mn(II) as a paramagnetic analogue of Mg(II) binding to plastocyanin. ► PRE-derived structure of the complex between plastocyanin and hexaquo Mn(II). ► Mg(II), a plausible modulator of electron donation to photosystem 1.
Keywords: EPR; paramagnetic relaxation enhancement; photosynthesis; plastocyanin; protein-protein interaction; PsaF;

High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units by Kersti Tepp; Igor Shevchuk; Vladimir Chekulayev; Natalja Timohhina; Andrey V. Kuznetsov; Rita Guzun; Valdur Saks; Tuuli Kaambre (1549-1561).
The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C vi JATP ) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C vi JATP were for system A: Complex I - 0.19, Complex III – 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) – 0.11, ATP synthase – 0.01, Pi carrier - 0.20, and the sum of C vi JATP was 0.75. In the presence of 10 mM creatine (system B) the C vi JATP were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C vi JATP were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C vi JATP was 1.33 and 3.84, respectively. Thus, C vi JATP were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.► We studied the distribution of the control between the components of MI supercomplex. ► The flux control coefficients were measured using two protocols. ► One protocol was with direct ADP activation and the second with creatine activation. ► We showed that the metabolic control is stronger in the case of creatine activation. ► In physiological conditions the energy transfer is regulated by the MI supercomplex.
Keywords: Permeabilized cardiac cells; Respiration; Heart; Mitochondria; Creatine kinase; Metabolic Control Analysis;

Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria by Ricardo Lagoa; Ilaria Graziani; Carmen Lopez-Sanchez; Virginio Garcia-Martinez; Carlos Gutierrez-Merino (1562-1572).
Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H2O2 production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H2O2 production by mitochondria from both tissues (IC50  ≈ 1–2 μM), even when H2O2 production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.Display Omitted► Flavonoids have different effects on H2O2 production by heart and brain mitochondria. ► Quercetin, kaempferol and epicatechin inhibit mitochondrial H2O2 production. ► Quercetin, kaempferol and apigenin inhibit Complex I activity. ► Epicatechin, quercetin and kaempferol reduce cytochrome c.
Keywords: Flavonoid; Mitochondria; Hydrogen peroxide production; Complex I; Ubiquinone; Cytochrome c;

Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes by Blake R. Zelickson; Gloria A. Benavides; Michelle S. Johnson; Balu K. Chacko; Aparna Venkatraman; Aimee Landar; Angela M. Betancourt; Shannon M. Bailey; Victor M. Darley-Usmar (1573-1582).
Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (•NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since •NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to •NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS−/− mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS−/− mice suggesting that increased mitochondrial sensitivity to •NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of •NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.► Mitochondrial damage and hypoxia is important in alcohol-dependent hepatotoxicity. ► Cellular bioenergetic reserve capacity is decreased by chronic ethanol exposure. ► Lower reserve capacity increases the sensitivity of cells to nitric oxide and hypoxia. ► Nitric oxide and hypoxic stress lower bioenergetics in response to ethanol in vivo.
Keywords: Ethanol; Inducible nitric oxide synthase; Bioenergetic; Reserve capacity;

Low temperature FTIR spectroscopy provides new insights in the pH-dependent proton pathway of proteorhodopsin by Mirka-Kristin Verhoefen; Gabriela Schäfer; Sarika Shastri; Ingrid Weber; Clemens Glaubitz; Werner Mäntele; Josef Wachtveitl (1583-1590).
In the presented study the low pH photocycle of proteorhodopsin is extensively investigated by means of low temperature FTIR spectroscopy. Besides the already well-known characteristics of the all-trans and 13-cis retinal vibrations the 77 K difference spectrum at pH 5.1 shows an additional negative signal at 1744 cm−1 which is interpreted as indicator for the L state. The subsequent photocycle steps are investigated at temperatures higher than 200 K. The combination of visible and FTIR spectroscopy enabled us to observe that the deprotonation of the Schiff base is linked to the protonation of an Asp or Glu side chain — the new proton acceptor under acidic conditions. The difference spectra of the late intermediates are characterized by large amide I changes and two further bands ((−)1751 cm−1  / (+)1725 cm-1) in the spectral region of the Asp/Glu ν(C = O) vibrations. The band position of the negative signature points to a transient deprotonation of Asp-97. In addition, the pH dependence of the acidic photocycle was investigated. The difference spectra at pH 5.5 show distinct differences connected to changes in the protonation state of key residues. Based on our data we propose a three-state model that explains the complex pH dependence of PR.Display Omitted► Photocycle study of proteorhodopsin (PR) using low temperature FTIR spectroscopy. ► At pH 5.1 proper isomerization, large amide changes and an altered proton acceptor. ► At pH 5.5 changes concerning the backbone movements and the protonation steps. ► Proposal of a three-state model that explains the complex pH dependence of PR.
Keywords: Proteorhodopsin; Photocycle; pH dependence; FTIR spectroscopy; Low temperature;

A kinetic model of non-photochemical quenching in cyanobacteria by Maxim Y. Gorbunov; Fedor I. Kuzminov; Victor V. Fadeev; John Dongun Kim; Paul G. Falkowski (1591-1599).
High light poses a threat to oxygenic photosynthetic organisms. Similar to eukaryotes, cyanobacteria evolved a photoprotective mechanism, non-photochemical quenching (NPQ), which dissipates excess absorbed energy as heat. An orange carotenoid protein (OCP) has been implicated as a blue-green light sensor that induces NPQ in cyanobacteria. Discovered in vitro, this process involves a light-induced transformation of the OCP from its dark, orange form (OCPo) to a red, active form, however, the mechanisms of NPQ in vivo remain largely unknown. Here we show that the formation of the quenching state in vivo is a multistep process that involves both photoinduced and dark reactions. Our kinetic analysis of the NPQ process reveals that the light induced conversion of OCPo to a quenching state (OCPq) proceeds via an intermediate, non-quenching state (OCPi), and this reaction sequence can be described by a three-state kinetic model. The conversion of OCPo to OCPi is a photoinduced process with the effective absorption cross section of 4.5 × 10− 3  Å2 at 470 nm. The transition from OCPi to OCPq is a dark reaction, with the first order rate constant of ~ 0.1 s− 1 at 25 °C and the activation energy of 21 kcal/mol. These characteristics suggest that the reaction rate may be limited by cis-trans proline isomerization of Gln224–Pro225 or Pro225–Pro226, located at a loop near the carotenoid. NPQ decreases the functional absorption cross-section of Photosystem II, suggesting that formation of the quenched centers reduces the flux of absorbed energy from phycobilisomes to the reaction centers by ~ 50%.► Formation of quenching centers involves light and dark reactions. ► The reaction sequence can be described by a three-state kinetic model. ► Rate of formation of the quenching centers is limited by prolyl isomerization. ► NPQ reduces the flux of energy from phycobilisomes to reaction centers by 50%.
Keywords: Cyanobacterium; Non-photochemical quenching; Orange carotenoid protein; Photosystem II; Synechocystis; Synechococcus;

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide by Alessandra Zulian; Justina Šileikytė; Valeria Petronilli; Sergio Bova; Federica Dabbeni-Sala; Gabriella Cargnelli; David Rennison; Margaret A. Brimble; Brian Hopkins; Paolo Bernardi; Fernanda Ricchelli (1600-1605).
We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18 kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.►The permeability transition pore (PTP) is an inner membrane mitochondrial channel. ►The rat-selective toxicant norbormide (NRB) is species-selective for PTP opening. ►The PTP-inducing effect is lost in mitoplasts (devoid of the outer membrane). ►NRB transport depends on the outer membrane protein TSPO. ►We have tentatively identified the TSPO residues responsible for transport of NRB.
Keywords: Mitochondria; Mitoplast; Permeability transition; Norbormide; TSPO;

X-ray structure of the dimeric cytochrome bc1 complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution by Thomas Kleinschroth; Michela Castellani; Chi H. Trinh; Nina Morgner; Bernhard Brutschy; Bernd Ludwig; Carola Hunte (1606-1615).
The respiratory cytochrome bc 1 complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c 1 changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-Å resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c 1 shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c 1 surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c 1 and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c 552, the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding.► First structure of fully active bacterial complex III at 2.7-Å resolution. ► Change of oligomerization state by truncation of acidic N-terminus of cytochrome c 1. ► Comparison of complex III structures reveals structural differences in cytochrome c 1. ► Model for the electron transfer complex with the native substrate cytochrome c 552.
Keywords: Respiratory complex III; Rhodobacteraceae; Stigmatellin; LILBID; Membrane protein structure;

Nitration of tyrosines 46 and 48 induces the specific degradation of cytochrome c upon change of the heme iron state to high-spin by Irene Díaz-Moreno; José M. García-Heredia; Antonio Díaz-Quintana; Miguel Teixeira; Miguel A. De la Rosa (1616-1623).
The Reactive Nitrogen and Oxygen Species (the so-called RNOS), which are well-known radicals formed in the mitochondria under nitro-oxidative cell stress, are responsible for nitration of tyrosines in a wide variety of proteins and, in particular, in cytochrome c (Cc). Only three out of the five tyrosine residues of human Cc, namely those at positions 67, 74 and 97, have been detected in vivo as nitrotyrosines. However, nitration of the two other tyrosines, namely those at positions 46 and 48, has never been detected in vivo despite they are both well-exposed to solvent. Here we investigate the changes in heme coordination and alkaline transition, along with the peroxidase activity and in cell degradation of Cc mutants in which all their tyrosine residues – with the only exception of that at position 46 or 48 – are replaced by phenylalanines. In Jurkat cell extracts devoid of proteases inhibitors, only the high-spin iron nitrated forms of these monotyrosine mutants are degraded. Altogether the resulting data suggest that nitration of tyrosines 46 and 48 makes Cc easily degradable upon turning the heme iron state to high-spin.► Cytochrome c nitrated at tyrosine 46 or 48 exhibits an enhanced peroxidase activity. ► Alkaline transition of nitrated cytochrome c becomes biologically relevant. ► The high-spin, nitrated species of cytochrome c are degraded in vivo.
Keywords: Apoptosis; High-spin heme iron; Peroxidase activity; Post-translational modification; Reactive nitrogen and oxygen species;

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits by Cindy E.J. Dieteren; Peter H.G.M. Willems; Herman G. Swarts; Jack Fransen; Jan A.M. Smeitink; Werner J.H. Koopman; Leo G.J. Nijtmans (1624-1633).
Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4–AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.► Chloramphenicol as a tool to impair mitochondrial translation. ► Complex I assembly dynamics during defective mitochondrial translation. ► Mitochondrial complex I is relatively immobile. ► Chloramphenicol differently affects live-cell dynamics of complex I subunits and assembly factors.
Keywords: Doxycycline; Mitochondria; FRAP; AcGFP1; Fluorescence gel analysis; Chloramphenicol;

Bacterial physiological responses integrate energy-coupling processes at the membrane level with metabolic energy demand. The regulatory design behind these responses remains largely unexplored. Propionigenium modestum is an adequate organism to study these responses because it presents the simplest scheme known integrating membrane potential generation and metabolic ATP consumption. A hypothetical sodium leak is added to the scheme as the sole regulatory site. Allosteric regulation is assumed to be absent. Information of the rate equations is not available. However, relevant features of the patterns of responses may be obtained using Metabolic Control Analysis (MCA) and Metabolic Control Design (MCD). With these tools, we show that membrane potential disturbances can be compensated by adjusting the leak flux, without significant perturbations of ATP consumption. Perturbations of membrane potential by ATP demand are inevitable and also require compensatory changes in the leak. Numerical simulations were performed with a kinetic model exhibiting the responses for small changes obtained with MCA and MCD. A modest leak (10% of input) was assumed for the reference state. We found that disturbances in membrane potential and ATP consumption, produced by environmental perturbations of the cation concentration, may be reverted to the reference state adjusting the leak. Leak changes can also compensate for undesirable effects on membrane potential produced by changes in nutrient availability or ATP demand, in a wide range of values. The system is highly robust to parameter fluctuations. The regulatory role of energy dissipating processes and the trade-off between energetic efficiency and regulatory capacity are discussed.► We model a scheme integrating membrane potential generation and ATP consumption. ► We examine if these two cellular processes can be independently regulated. ► Fluctuations in sodium gradient, nutrient and metabolic demand are considered. ► Resulting membrane potential perturbations may be corrected adjusting sodium leak. ► The system is robust to parameter fluctuations.
Keywords: Regulatory design; Membrane potential; Metabolic control; Energy coupling; Cation leak;

Structural elements of the mitochondrial preprotein-conducting channel Tom40 dissolved by bioinformatics and mass spectrometry by Dennis Gessmann; Nadine Flinner; Jens Pfannstiel; Andrea Schlösinger; Enrico Schleiff; Stephan Nussberger; Oliver Mirus (1647-1657).
Most mitochondrial proteins are imported into mitochondria from the cytosolic compartment. Proteins destined for the outer or inner membrane, the inter-membrane space, or the matrix are recognized and translocated by the TOM machinery containing the specialized protein import channel Tom40. The latter is a protein with β-barrel shape, which is suggested to have evolved from a porin-type protein. To obtain structural insights in the absence of a crystal structure the membrane topology of Tom40 from Neurospora crassa was determined by limited proteolysis combined with mass spectrometry. The results were interpreted on the basis of a structural model that has been generated for NcTom40 by using the structure of mouse VDAC-1 as a template and amino acid sequence information of ~ 270 different Tom40 and ~ 480 VDAC amino acid sequences for refinement. The model largely explains the observed accessible cleavage sites and serves as a structural basis for the investigation of physicochemical properties of the ensemble of our Tom40 sequence data set. By this means we discovered two conserved polar slides in the pore interior. One is possibly involved in the positioning of a pore-inserted helix; the other one might be important for mitochondrial pre-sequence peptide binding as it is only present in Tom40 but not in VDAC proteins. The outer surface of the Tom40 barrel reveals two conserved amino acid clusters. They may be involved in binding other components of the TOM complex or bridging components of the TIM machinery of the mitochondrial inner membrane.Display Omitted►Phylogenetic and structural insights into Tom40. ►By biochemical and bioinformatic means Tom40 is assigned as 19-stranded β-barrel. ►Intra-pore helix is positioned by a conserved helix anchor region at the pore wall. ►Tom40-specific polar slide inside the pore maybe involved in pre-protein recognition. ►Conserved pore surface regions explain TOM assembly and TOM-TIM bridging mutants.
Keywords: Tom40; VDAC; β-barrel; Eukaryotic porin; Mitochondria; Protein translocation;

Pure forms of the singlet oxygen sensors TEMP and TEMPD do not inhibit Photosystem II by Éva Hideg; Zsuzsanna Deák; Marja Hakala-Yatkin; Maarit Karonen; A. William Rutherford; Esa Tyystjärvi; Imre Vass; Anja Krieger-Liszkay (1658-1661).
In a recent article (Hakala-Yatkin and Tyystjärvi BBA 1807 (2011) 243–250) it was reported that the singlet oxygen spin traps 2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidone (TEMPD) inhibit Photosystem II (PSII), the water oxidizing enzyme. O2 evolution, chlorophyll fluorescence and thermoluminescence were measured and were shown to be greatly affected by these chemicals. This work casts doubts over an earlier body of work in which these chemicals were used as spin traps for monitoring 1O2 production when PSII was inhibited by high light intensities. Here we show that these spin probes hardly affect PSII. We show that the commercial batches of TEMPD and TEMP used by Hakala-Yatkin and Tyystjärvi contained impurities and/or derivatives that inhibited PSII and caused the specific effects on fluorescence. Earlier work that used pure spin traps to measure 1O2 during photoinhibition, thus remains valid. However, concern must be expressed towards using these spin traps without proper controls.► The pure spin traps TEMP and TEMPD do not inhibit photosystem II. ► Proper controls are necessary using these spin traps. ► The observations published in BBA 1807 (2011) 243-250 were critically analyzed.
Keywords: Singlet oxygen; Spin trap; EPR spectroscopy; Photosystem II;