BBA - Bioenergetics (v.1777, #7-8)

It is more than forty years since Peter Mitchell published his first ‘little grey book’ laying out his chemiosmotic hypothesis. Although ideas about the molecular mechanisms of the proton pumps have evolved considerably since then, his concept of ‘coupling through proton circuits’ remains remarkably prescient, and has provided the inspiration for the research careers of this author and many others. This review is a personal account of how the proton circuit has been followed from the little grey book, via brown fat and calcium transport to investigations into the life and death of neurons, Hercule Poirot's ‘little grey cells’.
Keywords: Mitochondria; Protons; Calcium; Brown fat; Membrane potential; Uncoupling;

Sorting and assembly of mitochondrial outer membrane proteins by Thomas Becker; F.-Nora Vögtle; Diana Stojanovski; Chris Meisinger (557-563).
In the last years the picture of protein import into the mitochondria has become much more complicated in terms of new components and new sorting pathways. These novel findings have also changed views concerning the biogenesis pathway of mitochondrial outer membrane proteins. In addition to proteins anchored with transmembrane α-helices, the endosymbiotic origin of the mitochondria has resulted in the presence of transmembrane β-barrels in this compartment. The sorting and assembly pathway of outer membrane proteins involves three machineries: the translocase of the outer membrane (TOM complex) the sorting and assembly machinery (SAM complex) and the MDM complex (mitochondrial distribution and morphology). Here we review recent developments on the biogenesis pathways of outer membrane proteins with a focus on Tom proteins, the most intensively studied class of these precursor proteins.
Keywords: Protein import; Assembly; SAM; TOM; Morphology;

A strikingly large number of mitochondrial DNA (mtDNA) mutations have been found to be the cause of respiratory chain and oxidative phosphorylation defects. These mitochondrial disorders were the first to be investigated after the small mtDNA had been sequenced in the 80s. Only recently numerous diseases resulting from mutations in nuclear genes encoding mitochondrial proteins have been characterized. Among these, nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of metabolites across the mitochondrial membrane. Mutations of mitochondrial carrier genes involved in mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine carrier deficiency, HHH syndrome, aspartate/glutamate isoform 2 deficiency, Amish microcephaly, and neonatal myoclonic epilepsy; these disorders are characterized by specific metabolic dysfunctions, depending on the physiological role of the affected carrier in intermediary metabolism. Defects of mitochondrial carriers that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phosphate and ADP, are responsible for diseases characterized by defective energy production. Herein, all the mitochondrial carrier-associated diseases known to date are reviewed for the first time. Particular emphasis is given to the molecular basis and pathogenetic mechanism of these inherited disorders.
Keywords: Carrier deficiencies; Mitochondria; Mitochondrial carriers; Mitochondrial diseases; Mitochondrial carrier-related diseases; Transporters;

Determination of torque generation from the power stroke of Escherichia coli F1-ATPase by Tassilo Hornung; Robert Ishmukhametov; David Spetzler; James Martin; Wayne D. Frasch (579-582).
The torque generated by the power stroke of Escherichia coli F1-ATPase was determined as a function of the load from measurements of the velocity of the γ-subunit obtained using a 0.25 µs time resolution and direct measurements of the drag from 45 to 91 nm gold nanorods. This result was compared to values of torque calculated using four different drag models. Although the γ-subunit was able to rotate with a 20× increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63 ± 8 pN nm, independent of the load on the enzyme.
Keywords: F1-ATPase; Gold nanorod; Molecular motor; High-speed data acquisition; Single molecule;

Probing the functional tolerance of the b subunit of Escherichia coli ATP synthase for sequence manipulation through a chimera approach by Yumin Bi; Joel C. Watts; Pamela Krauss Bamford; Lee-Ann K. Briere; Stanley D. Dunn (583-591).
A dimer of 156-residue b subunits forms the peripheral stator stalk of eubacterial ATP synthase. Dimerization is mediated by a sequence with an unusual 11-residue (hendecad) repeat pattern, implying a right-handed coiled coil structure. We investigated the potential for producing functional chimeras in the b subunit of Escherichia coli ATP synthase by replacing parts of its sequence with corresponding regions of the b subunits from other eubacteria, sequences from other polypeptides having similar hendecad patterns, and sequences forming left-handed coiled coils. Replacement of positions 55–110 with corresponding sequences from Bacillus subtilis and Thermotoga maritima b subunits resulted in fully functional chimeras, judged by support of growth on nonfermentable carbon sources. Extension of the T. maritima sequence N-terminally to position 37 or C-terminally to position 124 resulted in slower but significant growth, indicating retention of some capacity for oxidative phosphorylation. Portions of the dimerization domain between 55 and 95 could be functionally replaced by segments from two other proteins having a hendecad pattern, the distantly related E subunit of the Chlamydia pneumoniae V-type ATPase and the unrelated Ag84 protein of Mycobacterium tuberculosis. Extension of such sequences to position 110 resulted in loss of function. None of the chimeras that incorporated the leucine zipper of yeast GCN4, or other left-handed coiled coils, supported oxidative phosphorylation, but substantial ATP-dependent proton pumping was observed in membrane vesicles prepared from cells expressing such chimeras. Characterization of chimeric soluble b polypeptides in vitro showed their retention of a predominantly helical structure. The T. maritima b subunit chimera melted cooperatively with a midpoint more than 20 °C higher than the normal E. coli sequence. The GCN4 construct melted at a similarly high temperature, but with much reduced cooperativity, suggesting a degree of structural disruption. These studies provide insight into the structural and sequential requirements for stator stalk function.
Keywords: ATP synthase; b subunit; Chimeric protein; Coiled coil; Right-handed coiled coil; Hendecad pattern;

Structural organization of mitochondrial ATP synthase by Ilka Wittig; Hermann Schägger (592-598).
Specific modules and subcomplexes like F1 and F0-parts, F1-c subcomplexes, peripheral and central stalks, and the rotor part comprising a ring of c-subunits with attached subunits γ, δ, and ε can be identified in yeast and mammalian ATP synthase. Four subunits, α3β3, OSCP, and h, seem to form a structural entity at the extramembranous rotor/stator interface (γ/α3β3) to hold and stabilize the rotor in the holo-enzyme. The intramembranous rotor/stator interface (c-ring/a-subunit) must be dynamic to guarantee unhindered rotation. Unexpectedly, a c10a-assembly could be isolated with almost quantitive yield suggesting that an intermediate step in the rotating mechanism was frozen under the conditions used. Isolation of dimeric a-subunit and (c10)2a2-complex from dimeric ATP synthase suggested that the a-subunit stabilizes the same monomer–monomer interface that had been shown to involve also subunits e, g, b, i, and h. The natural inhibitor protein Inh1 does not favor oligomerization of yeast ATP synthase. Other candidates for the oligomerization of dimeric ATP synthase building blocks are discussed, e.g. the transporters for inorganic phosphate and ADP/ATP that had been identified as constituents of ATP synthasomes. Independent approaches are presented that support previous reports on the existence of ATP synthasomes in the mitochondrial membrane.
Keywords: ATP synthase; Supramolecular organization; Protein–protein interaction; Cristae membrane; Mitochondria;

Structure and regulation of the vacuolar ATPases by Daniel J. Cipriano; Yanru Wang; Sarah Bond; Ayana Hinton; Kevin C. Jefferies; Jie Qi; Michael Forgac (599-604).
The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for both acidification of intracellular compartments and, for certain cell types, proton transport across the plasma membrane. Intracellular V-ATPases function in both endocytic and intracellular membrane traffic, processing and degradation of macromolecules in secretory and digestive compartments, coupled transport of small molecules such as neurotransmitters and ATP and in the entry of pathogenic agents, including envelope viruses and bacterial toxins. V-ATPases are present in the plasma membrane of renal cells, osteoclasts, macrophages, epididymal cells and certain tumor cells where they are important for urinary acidification, bone resorption, pH homeostasis, sperm maturation and tumor cell invasion, respectively. The V-ATPases are composed of a peripheral domain (V1) that carries out ATP hydrolysis and an integral domain (V0) responsible for proton transport. V1 contains eight subunits (A–H) while V0 contains six subunits (a, c, c′, c″, d and e). V-ATPases operate by a rotary mechanism in which ATP hydrolysis within V1 drives rotation of a central rotary domain, that includes a ring of proteolipid subunits (c, c′ and c″), relative to the remainder of the complex. Rotation of the proteolipid ring relative to subunit a within V0 drives active transport of protons across the membrane. Two important mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V1 and V0 domains and changes in coupling efficiency of proton transport and ATP hydrolysis. This review focuses on recent advances in our lab in understanding the structure and regulation of the V-ATPases.
Keywords: Vacuolar ATPases; ATP-dependent proton transport; V-ATPase structure; Regulation of V-ATPase activity; Function of intracellular and plasma membrane V-ATPases;

Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments by Benjamin Varco-Merth; Raimund Fromme; Meitian Wang; Petra Fromme (605-612).
The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10–15 c-subunits is commonly thought to drive rotation of the rotor moiety (c10–14γɛ) relative to stator moiety (α3β3δab2). Here we report the isolation and crystallization of the c14-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 Å. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase.
Keywords: ATP synthase; Crystallization; Membrane proteins; Chlorophyll; Carotenoid;

Structure, function and interactions of the PufX protein by Kate Holden-Dye; Lucy I. Crouch; Michael R. Jones (613-630).
The PufX protein is an important component of the reaction centre–light-harvesting 1 (RC–LH1) complex of Rhodobacter species of purple photosynthetic bacteria. Early studies showed that removal of the PufX protein causes changes in the structure of the RC–LH1 complex that result in a loss of the capacity for photosynthetic growth, and that this loss can be overcome though further mutations that change the structure of the LH1 antenna. More recent studies have examined interactions of the PufX protein with other components of the RC–LH1 complex. This review considers our current understanding of the structure and function of the PufX protein, how this protein interacts with other components of the photosynthetic membrane, and its influence on the oligomeric state of the RC–LH1 complex and the larger-scale architecture of the photosynthetic membrane.
Keywords: Photosynthesis; Purple bacteria; PufX; Light-harvesting; Photosystem;

The 2-methoxy group of ubiquinone is essential for function of the acceptor quinones in reaction centers from Rba. sphaeroides by Colin A. Wraight; Ahmet S. Vakkasoglu; Yuri Poluektov; Aidas J. Mattis; Danielle Nihan; Bruce H. Lipshutz (631-636).
The orientation of a methoxy substituent is known to substantially influence the electron affinity and vibrational spectroscopy of benzoquinones, and has been suggested to be important in determining the function of ubiquinone as a redox cofactor in bioenergetics. Ubiquinone functions as both the primary (QA) and secondary (QB) quinone in the reaction centers of many purple photosynthetic bacteria, and is almost unique in its ability to establish the necessary redox free energy gap for 1-electron transfer between them. The role of the methoxy substitution in this requirement was examined using monomethoxy analogues of ubiquinone-4 — 2-methoxy-3,5-dimethyl-6-isoprenyl-1,4-benzoquinone (2-MeO-Q) and 3-methoxy-2,5-dimethyl-6-isoprenyl-1,4-benzoquinone (3-MeO-Q). Only 2-MeO-Q was able to simultaneously act as QA and QB and the necessary redox potential tuning was shown to occur in the QB site. In the absence of active QB, the IR spectrum of the monomethoxy quinones was examined in vitro and in the QA site, and a novel distinction between the two methoxy groups was tentatively identified, consistent with the unique role of the 2-methoxy group in distinguishing QA and QB functionality.
Keywords: Quinone; Reaction center; Infrared spectroscopy; Electron transfer; Rba. sphaeroides;

An ancient look at UCP1 by Martin Klingenspor; Tobias Fromme; David A. Hughes; Lars Manzke; Elias Polymeropoulos; Tobias Riemann; Magdalene Trzcionka; Verena Hirschberg; Martin Jastroch (637-641).
Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure–function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.
Keywords: Mitochondria; Brown adipose tissue; Nonshivering thermogenesis; Mitochondrial transporters;

Within brown-fat cells, UCP1-mediated fatty acid-induced uncoupling is independent of fatty acid metabolism by Irina G. Shabalina; Emma C. Backlund; Jacob Bar-Tana; Barbara Cannon; Jan Nedergaard (642-650).
In the present investigation, we have utilized the availability of UCP1(−/−) mice to examine a wide range of previously proposed lipid activators of Uncoupling Protein 1 (UCP1) in its native environment, i.e. in the brown-fat cells. A non-metabolizable fatty acid analogue, β,β¢-methyl-substituted hexadecane α,ω-dicarboxylic acid (Medica-16) is a potent UCP1 (re)activator in brown-fat cells, despite its bipolar structure. All-trans-retinoic acid activates UCP1 within cells, whereas β-carotene only does so after metabolism. The UCP1-dependent effects of fatty acids are positively correlated with their chain length. Medium-chain fatty acids are potent UCP1 activators in cells, despite their lack of protonophoric properties in mitochondrial membranes. Thus, neither the ability to be metabolized nor an innate uncoupling/protonophoric ability is a necessary property of UCP1 activators within brown-fat cells.
Keywords: Brown-fat cell; Mitochondrial uncoupling; All-trans-retinoic acid; Medium-chain fatty acid; Medica-16; Short-chain fatty acid; Carotene;

A novel potassium channel in skeletal muscle mitochondria by Jolanta Skalska; Marta Piwońska; Elzbieta Wyroba; Liliana Surmacz; Rafal Wieczorek; Izabela Koszela-Piotrowska; Joanna Zielińska; Piotr Bednarczyk; Krzysztof Dołowy; Grzegorz M. Wilczynski; Adam Szewczyk; Wolfram S. Kunz (651-659).
In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca2+ was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca2+ were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BKCa channel). Furthermore, NS1619, a BKCa-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BKCa channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K+ transport by opening of the BKCa channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.
Keywords: Mitochondria; Potassium channel; Skeletal muscle;

Exploring the inhibitor binding pocket of respiratory complex I by Uta Fendel; Maja A. Tocilescu; Stefan Kerscher; Ulrich Brandt (660-665).
Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I 50 values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron–sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C12E8 (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.
Keywords: Complex I; Mitochondria; Ubiquinone; Inhibitor; Rotenone; DQA; C12E8; Mutagenesis; Yarrowia lipolytica;

MITOCHIP assessment of differential gene expression in the skeletal muscle of Ant1 knockout mice: Coordinate regulation of OXPHOS, antioxidant, and apoptotic genes by Vaidya Subramaniam; Pawel Golik; Deborah G. Murdock; Shawn Levy; Keith W. Kerstann; Pinar E. Coskun; Goarik A. Melkonian; Douglas C. Wallace (666-675).
Genetic inactivation of the nuclear-encoded mitochondrial heart-muscle adenine nucleotide translocator-1 (ANT1), which exports mitochondrial ATP to the cytosol in both humans (ANT1−/−) and mice (Ant1−/−), results in lactic acidosis and mitochondrial cardiomyopathy and myopathy, the latter involving hyper-proliferation of mitochondria, induction of oxidative phosphorylation (OXPHOS) enzymes, increased reactive oxygen species (ROS), and excessive mtDNA damage. To understand these manifestations, we analyzed Ant1−/− mouse skeletal muscle for changes in gene expression using our custom 644 and 1087 gene MITOCHIP microarrays and for changes in the protein levels of key mitochondrial transcription factors. Thirty-four mRNAs were found to be up-regulated and 29 mRNAs were down-regulated. Up-regulated mRNAs included the mitochondrial DNA (mtDNA) polypeptide and rRNA genes, selected nuclear-encoded OXPHOS genes, and stress-response genes including Mcl-1. Down-regulated mRNAs included glycolytic genes, pro-apoptotic genes, and c-Myc. The mitochondrial regulatory proteins Pgc-1α, Nrf-1, Tfam, and myogenin were up-regulated and could account for the induction of the OXPHOS and antioxidant enzymes. By contrast, c-Myc levels were reduced and might account for a reduction in apoptotic potential. Therefore, the Ant1−/− mouse skeletal muscle demonstrates that energy metabolism, antioxidant defenses, and apoptosis form an integrated metabolic network.
Keywords: Mitochondria; Microarray; Energy deficiency; OXPHOS; Adenine nucleotide translocator; ANT1;

We have developed a new method to quantify the transmembrane electrochemical proton gradient present in chloroplasts of dark-adapted leaves. When a leaf is illuminated by a short pulse of intense light, we observed that the light-induced membrane potential changes, measured by the difference of absorption (520 nm–546 nm), reach a maximum value (~ 190 mV) determined by ion leaks that occur above a threshold level of the electrochemical proton gradient. After the light-pulse, the decay of the membrane potential follows a multiphasic kinetics. A marked slowdown of the rate of membrane potential decay occurs ~ 100 ms after the light-pulse, which has been previously interpreted as reflecting the switch from an activated to an inactivated state of the ATP synthase (Junge, W., Rumberg, B. and Schröder, H., Eur. J. Biochem. 14 (1970) 575–581). This transition occurs at ~ 110 mV, thereby providing a second reference level. On this basis, we have estimated the Δμ˜H+ level that pre-exists in the dark. Depending upon the physiological state of the leaf, this level varies from 40 to 70 mV. In the dark, the Δμ˜H+ collapses upon addition of inhibitors of the respiratory chain, thus showing that it results from the hydrolysis of ATP of mitochondrial origin. Illumination of the leaf for a period longer than several seconds induces a long-lived Δμ˜H+ increase (up to ~ 150 mV) that reflects the light-induced increase in ATP concentration. Following the illumination, Δμ˜H+ relaxes to its dark-adapted value according a multiphasic kinetics that is completed in more than 1 h. In mature leaf, the deactivation of the Benson–Calvin cycle follows similar kinetics as Δμ˜H+ decay, showing that its state of activation is mainly controlled by ATP concentration.
Keywords: ATP synthase; Electrochemical proton gradient; Membrane potential;

Xanthorhodopsin is a light-driven proton pump like bacteriorhodopsin, but made more effective for collecting light by its second chromophore, salinixanthin, a carotenoid. Action spectra for transport and fluorescence of the retinal upon excitation of the carotenoid indicate that the carotenoid functions as an antenna to the retinal. The calculated center-to-center distance and angle of the transition moments of the two chromophores are 11 Å and 56°, respectively. As expected from their proximity, the carotenoid and the retinal closely interact: tight binding of the carotenoid, as indicated by its sharpened vibration bands and intense induced circular dichroism in the visible, is removed by hydrolysis of the retinal Schiff base, and restored upon reconstitution with retinal. This antenna system, simpler than photosynthetic complexes, is well-suited to study features of excited-state energy migration.
Keywords: Xanthorhodopsin; Retinal protein; Antenna carotenoid; Energy transfer; Fluorescence anisotropy;

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of various neurological diseases and aging. But the exact sites of ROS generation in brain tissue remained so far elusive. Here, we provide direct experimental evidence that at least 50% of total ROS generation in succinate-oxidizing homogenates of brain tissue can be attributed to complex I of mitochondrial respiratory chain. Applying quantitative methods for ROS detection we observed in different preparations from human, rat and mouse brain (digitonin-permeabilized tissue homogenates and isolated mitochondria) a linear relationship between rate of oxygen consumption and ROS generation with succinate as mitochondrial substrate. This quantitative relationship indicates, that under the particular conditions of oxygen saturation about 1% of the corresponding respiratory chain electron flow is redirected to form superoxide. Since we observed in mouse and rat brain mitochondria a unique dependency of both forward and reverse electron flow-dependent mitochondrial H2O2 production on NAD redox state, we substantiated previous evidence that the FMN moiety of complex I is the major donor of electrons for the single electron reduction of molecular oxygen.
Keywords: Reactive oxygen species; Brain tissue; Mitochondria; Respiratory chain complex I; Respiratory chain complex III;

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory flavo-FeS complex composed of the six subunits NqrA–F. The Na+-NQR was produced as His6-tagged protein by homologous expression in V. cholerae. The isolated complex contained near-stoichiometric amounts of non-covalently bound FAD (0.78 mol/mol Na+-NQR) and riboflavin (0.70 mol/mol Na+-NQR), catalyzed NADH-driven Na+ transport (40 nmol Na+min− 1 mg− 1), and was inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide. EPR spectroscopy showed that Na+-NQR as isolated contained very low amounts of a neutral flavosemiquinone (10− 3 mol/mol Na+-NQR). Reduction with NADH resulted in the formation of an anionic flavosemiquinone (0.10 mol/mol Na+-NQR). Subsequent oxidation of the Na+-NQR with ubiquinone-1 or O2 led to the formation of a neutral flavosemiquinone (0.24 mol/mol Na+-NQR). We propose that the Na+-NQR is fully oxidized in its resting state, and discuss putative schemes of NADH-triggered redox transitions.
Keywords: Respiration; NADH dehydrogenase; Flavin; Na+ transport;

NADH-quinone oxidoreductase (complex I) in bovine heart mitochondria has a molecular weight of approximately 1 million Da composed of 45 distinct subunits. It is the largest energy transducing complex so far known. Bacterial complex I is simpler and smaller, but the essential redox components and the basic mechanisms of electron and proton translocation are the same. Over the past three decades, Ohnishi et al. have pursued extensive EPR studies near liquid helium temperatures and characterized most of the iron–sulfur clusters in complex I. Recently, Yakovlev et al. [G. Yakovlev, T. Reda, J. Hirst, Reevaluating the relationship between EPR spectra and enzyme structure for the iron-sulfur clusters in NADH:quinone oxidoreductase, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12720–12725] challenged Ohnishi's group by claiming that there were EPR “misassignments” among clusters N4, N5 and N6b (in order to prevent confusion, we used current consensus nomenclature, as the nickname). They claimed that we misassigned EPR signals arising from cluster N5 to cluster N4, and signals from cluster N6b to cluster N4. They also proposed that cluster N5 has (4Cys)-ligands. Based on the accumulated historical data and recent results of our site-specific mutagenesis experiments, we confirmed that cluster N5 has (1His + 3Cys)-ligands as we had predicted. We revealed that E. coli cluster N5 signals could be clearly detected at the sample temperature around 3 K with microwave power higher than 5 mW. Thus Hirst's group could not detect N5 signals under any of their EPR conditions, reported in their PNAS paper. It seems that they misassigned the signals from cluster N4 to N5. As to the claim of “misassignment” between clusters N4 and N6b, that was not a possibility because our mutagenesis systems did not contain cluster N6b. Therefore, we believe that we have not made any “misassignment” in our work.
Keywords: Complex I; Iron–sulfur cluster; EPR; Site-directed mutagenesis; NADH-Q oxidoreductase;

Complex I (NADH:ubiquinone oxidoreductase) is the largest protein complex of bacterial and mitochondrial respiratory chains. The first three-dimensional structure of bacterial complex I in vitrified ice was determined by electron cryo-microscopy and single particle analysis. The structure of the Escherichia coli enzyme incubated with either NAD+ (as a reference) or NADH was calculated to 35 and 39 Å resolution, respectively. The X-ray structure of the peripheral arm of Thermus thermophilus complex I was docked into the reference EM structure. The model obtained indicates that Fe–S cluster N2 is close to the membrane domain interface, allowing for effective electron transfer to membrane-embedded quinone. At the current resolution, the structures in the presence of NAD+ or NADH are similar. Additionally, side-view class averages were calculated for the negatively stained bovine enzyme. The structures of bovine complex I in the presence of either NAD+ or NADH also appeared to be similar. These observations indicate that conformational changes upon reduction with NADH, suggested to occur by a range of studies, are smaller than had been thought previously. The model of the entire bacterial complex I could be built from the crystal structures of subcomplexes using the EM envelope described here.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Membrane protein structure; Single particle analysis; Electron microscopy;

Mammalian complex I: A regulable and vulnerable pacemaker in mitochondrial respiratory function by Sergio Papa; Domenico De Rasmo; Salvatore Scacco; Anna Signorile; Zuzana Technikova-Dobrova; Giuseppe Palmisano; Anna Maria Sardanelli; Francesco Papa; Damiano Panelli; Raffaella Scaringi; Arcangela Santeramo (719-728).
In this paper the regulatory features of complex I of mammalian and human mitochondria are reviewed. In a variety of mitotic cell-line cultures, activation in vivo of the cAMP cascade, or direct addition of cAMP, promotes the NADH–ubiquinone oxidoreductase activity of complex I and lower the cellular level of ROS. These effects of cAMP are found to be associated with PKA-mediated serine phosphorylation in the conserved C-terminus of the subunit of complex I encoded by the nuclear gene NDUFS4. PKA mediated phosphorylation of this Ser in the C-terminus of the protein promotes its mitochondrial import and maturation. Mass-spectrometry analysis of the phosphorylation pattern of complex I subunits is also reviewed.
Keywords: Complex I; Proton pump; cAMP cascade; PKA; Mitochondrial import; Mitochondrial phosphoproteome;

The quantitative data on the binding affinity of NADH, NAD+, and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.
Keywords: Bioenergetics; Respiration; Mitochondria; Complex I; Nucleotide binding;

Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) by Daniel Schneider; Thomas Pohl; Julia Walter; Katerina Dörner; Markus Kohlstädt; Annette Berger; Volker Spehr; Thorsten Friedrich (735-739).
The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and the mitochondria of most eukaryotes. In general, the bacterial complex consists of 14 different subunits. In addition to the homologues of these subunits, the mitochondrial complex contains approximately 31 additional proteins. While it was shown that the mitochondrial complex is assembled from distinct intermediates, nothing is known about the assembly of the bacterial complex. We used Escherichia coli mutants, in which the nuo-genes coding the subunits of complex I were individually disrupted by an insertion of a resistance cartridge to determine whether they are required for the assembly of a functional complex I. No complex I-mediated enzyme activity was detectable in the mutant membranes and it was not possible to extract a structurally intact complex I from the mutant membranes. However, the subunits and the cofactors of the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of some of the nuo-mutants. It is discussed whether this fragment represents an assembly intermediate. In addition, a membrane-bound fragment exhibiting NADH/ferricyanide oxidoreductase activity and containing the iron–sulfur cluster N2 was detected in one mutant.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Assembly; nuo-genes; EPR spectroscopy; Escherichia coli;

Is supercomplex organization of the respiratory chain required for optimal electron transfer activity? by M.L. Genova; A. Baracca; A. Biondi; G. Casalena; M. Faccioli; A.I. Falasca; G. Formiggini; G. Sgarbi; G. Solaini; G. Lenaz (740-746).
The supra-molecular assembly of the main respiratory chain enzymatic complexes in the form of “supercomplexes” has been proved by structural and functional experimental evidence. This evidence strongly contrasts the previously accepted Random Diffusion Model stating that the complexes are functionally connected by lateral diffusion of small redox molecules (i.e. Coenzyme Q and cytochrome c).This review critically examines the available evidence and provides an analysis of the functional consequences of the intermolecular association of the respiratory complexes pointing out the role of Coenzyme Q and of cytochrome c as channeled or as freely diffusing intermediates in the electron transfer activity of their partner enzymes.
Keywords: Respiratory chain; Supercomplex; Coenzyme Q; Cytochrome c;

ATP-synthase assembly requires coordinated control of ATP mRNA translation; this may e.g. occur through the formation of mRNA–protein complexes. In this study we aim to identify sequences in the 3'UTR of the β-subunit F1-ATPase mRNA necessary for RNA–protein complex formation. We examined the interaction between a brain cytoplasmic protein extract and in vitro-synthesized β-subunit 3'UTR probes containing successive accumulative 5'- and 3'-deletions, as well as single subregion deletions, with or without poly(A) tail. Using electrophoretic mobility shift assays we found that two major RNA–protein complexes (here called RPC1 and RPC2) were formed with the full-length 3'UTR. The RPC2 complex formation was fully dependent on the presence of both the poly(A) tail and one subregion directly adjacent to it. For RPC1 complex formation, a 3'UTR sequence stretch (experimentally divided into three subregions) adjacent to but not including the poly(A) tail was necessary. This sequence stretch includes a conserved 40-nucleotide region that, according to the structure prediction program mfold, is able to fold into a characteristic stem–loop structure. Since the formation of the RPC1 complex was not dependent on a conventional sequence motif in the 3'UTR of the β-subunit mRNA but rather on the presence of the predicted stem–loop-forming region as such, we hypothetize that this RNA region, by forming a stem–loop in the 3'UTR β-subunit mRNA, is necessary for formation of the RNA–protein complex.
Keywords: RNA secondary structure; Gel mobility shift assay; Mitochondrial F0F1-ATPase; 3'untranslated region; Stem–loop; RNA–protein complexes;

Energetics of protein translocation into mitochondria by Dejana Mokranjac; Walter Neupert (758-762).
Biogenesis of mitochondria depends on the coordinated action of at least six protein translocases present in both mitochondrial membranes. They use different energy sources to drive unidirectional transport of proteins across and into mitochondrial membranes. Here we present an overview on the energetic requirements of different mitochondrial import pathways.
Keywords: Mitochondria; Driving force; Protein transport; Membrane potential; ATP; Oxidative folding;

Sepsis is a complex pathophysiological disorder arising from a systemic inflammatory response to infection. Patients are clinically classified according to the presence of signs of inflammation alone, multiple organ failure (MOF), or organ failure plus hypotension (septic shock). The organ damage that occurs in MOF is not a direct effect of the pathogen itself, but rather of the dysregulated inflammatory response of the patient. Although mechanisms underlying MOF are not completely understood, a disruption in cellular energetic metabolism is increasingly implicated. In this review, we describe how various factors affecting cellular ATP supply and demand appear to be altered in sepsis, and how these vary through the timecourse. We will emphasise the need for an integrated systems approach to determine the relative importance of these factors in both the failure and recovery of different organs. A modular framework is proposed that can be used to assess the control hierarchy of cellular energetics in this complex pathophysiological condition.
Keywords: Mitochondria; Sepsis; Inflammation; Multiple organ failure; Mitochondrial dysfunction; Top–down metabolic control; Cellular energetic metabolism;

Mitochondrial uncoupling protein 1 expression in thymocytes by Alison E. Adams; Audrey M. Carroll; Padraic G. Fallon; Richard K. Porter (772-776).
Using an antibody specific and selective to mitochondrial uncoupling protein 1 (UCP1) peptide, this study confirms the observation that UCP 1 is present in thymocytes isolated from UCP 1 wild-type, but not UCP 1 knock-out mice. UCP 1 is also shown to be present in thymocytes isolated from rat. It was also demonstrated that an antibody raised to the full-length UCP 1 protein appears to be non-specific for UCP 1, as it detects protein in UCP 1 wild-type and UCP 1 knock-out mice, protein in mitochondria isolated from brown adipose tissue of both UCP 1 wild-type and UCP 1 knock-out mice, as well as detecting protein in mitochondria isolated from rat spleen, kidney, skeletal muscle and liver, tissues that do not express UCP 1. We were also able to show that CIDEA, a soluble protein with a suggested role in regulating UCP 1 function, is equally abundant in thymocytes from UCP 1 wild-type and UCP 1 knock-out mice. Taken together our data demonstrate that (a) UCP 1 is present in rat and mouse thymocytes, (b) that the antibody to full-length UCP 1 is not specific for UCP 1 and (c) that the absence of UCP 1 does not affect native expression of CIDEA in thymocytes.
Keywords: Uncoupling protein-1; Thymus; Thymocytes; Mitochondria; UCP 1 knock-out mice; Confocal microscopy;

Complex I and energy thresholds in the brain by Rashmi U. Pathak; Gavin P. Davey (777-782).
Mitochondrial electron transport chain (ETC) deficiencies are thought to underlie defects in energy metabolism and have been implicated in the neurodegenerative process. In particular, reductions in complex I activities in Parkinson's disease are thought to cause bioenergetic dysfunction with subsequent degeneration of dopaminergic neurons. In terms of bioenergetics and assessing ETC-related problems in the brain, the presence of heterogeneous mitochondria has complicated matters as isolated non-synaptic mitochondria have different energy thresholds and flux control coefficients compared to isolated mitochondria of synaptic origin. The molecular mechanisms that underlie complex I deficiencies in the parkinsonian brain are unknown and are the source of intensive research. This review explores the relationship between complex I activity and energy metabolism in the brain as well as the nature of the complex I defect.
Keywords: Complex I; Brain mitochondria; Metabolic control analysis; Neurodegeneration; Parkinson's disease;

Age-related changes in H2O2 production and bioenergetics in rat brain synaptosomes by Seán M. Kilbride; Jayne E. Telford; Gavin P. Davey (783-788).
Detrimental changes to mitochondrial function have been shown to occur with age. In this study we examined the levels of H2O2 production, in situ mitochondrial membrane potential (Δψ m), oxygen consumption (JO2) and electron transport chain (ETC) enzyme activities in synaptosomes isolated from rats of two age groups, 6 and 18 months. The rate of H2O2 production in synaptosomes was found to be higher in the 18-month old group compared to that of 6-month old. Δψ m was found to be significantly lower in synaptosomes from the older rats, which also correlated with a reduction in JO2. Measurement of the individual electron transport chain enzyme activities revealed that reduced complex II/III and complex IV activities were the possible contributors to the reduced bioenergetic function in synaptosomes from the older rats. These data suggest that ageing may lead to increased nerve terminal H2O2 production while simultaneous deleterious effects on bioenergetic function occur in in situ synaptosomal mitochondria. In addition, Ca2+-independent glutamate release was found to be increased at lower levels of complex I inhibition in the synaptosomes from older rats, suggesting that reduction of mitochondrial function may potentiate excitotoxic conditions in the ageing brain.
Keywords: Synaptosomes; Mitochondria; Ageing; Reactive oxygen species; Complex 1;

Regulation of glycolysis and pentose–phosphate pathway by nitric oxide: Impact on neuronal survival by Juan P. Bolaños; Maria Delgado-Esteban; Angel Herrero-Mendez; Seila Fernandez-Fernandez; Angeles Almeida (789-793).
Besides its essential role at regulating neural functions through cyclic GMP, nitric oxide is emerging as an endogenous physiological modulator of energy conservation for the brain. Thus, nitric oxide inhibits cytochrome c oxidase activity in neurones and glia, resulting in down-regulation of mitochondrial energy production. The subsequent increase in AMP facilitates the activation of 5′-AMP-dependent protein kinase, which rapidly triggers the activation of 6-phosphofructo-1-kinase – the master regulator of the glycolytic pathway – and Glut1 and Glut3 — the main glucose transporters in the brain. In addition, nitric oxide activates glucose-6-phosphate dehydrogenase, the first and rate-limiting step of the pentose–phosphate pathway. Here, we review recent evidences suggesting that nitric oxide exerts a fine control of neuronal energy metabolism by tuning the balance of glucose-6-phosphate consumption between glycolysis and pentose–phosphate pathway. This may have important implications for our understanding of the mechanisms controlling neuronal survival during oxidative stress and bioenergetic crisis.
Keywords: Mitochondria; Pentose–phosphate pathway; 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; AMP-activated protein kinase; Neurodegeneration; Oxidative stress;

Oxidative stress and subsequent impairment of mitochondrial function is implicated in the neurodegenerative process and hence in diseases such as Parkinson's and Alzheimer's disease. Within the brain, neuronal and astroglial cells can display a differential susceptibility to oxidant exposure. Thus, astrocytes can up regulate glutathione availability and, in response to mitochondrial damage, glycolytic flux. Whilst neuronal cells do not appear to possess such mechanisms, neuronal glutathione status may be enhanced due to the trafficking of glutathione precursors from the astrocyte. However, when antioxidants reserves are not sufficient or the degree of oxidative stress is particularly great, mitochondrial damage occurs, particularly at the level of complex IV (cytochrome oxidase). Whilst the exact mechanism for the loss of activity of this enzyme complex is not know, it is possible that loss and/or oxidative modification of the phospholipid, cardiolipin is a critical factor. Consequently, in this short article, we also consider (a) cardiolipin metabolism and function, (b) the susceptibility of this molecule to undergo oxidative modification following exposure to oxidants such as peroxynitrite, (c) loss of mitochondrial cardiolipin in neurodegenerative disorders, (d) methods of detecting cardiolipin and (e) possible therapeutic strategies that may protect cardiolipin from oxidative degradation.
Keywords: Oxidative Stress; Mitochondria; Neurodegeneration; Nitric Oxide; Glutathione; Cardiolipin;

H2O2 generation is decreased by calcium in isolated brain mitochondria by Zsofia Komary; Laszlo Tretter; Vera Adam-Vizi (800-807).
Release of H2O2 in response to Ca2+ loads (1–100 μM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca2+ (10 μM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H2O2 release, taken as an indication of H2O2 production, was decreased by 21.8 ± 1.6% in the presence of NADH-linked substrates and by 86.5 ± 1.8% with succinate. Parallel with this, a Ca2+-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H2O2 release from mitochondria was decreased, but Ca2+ no longer influenced the generation of H2O2. We suggest that the decreased H2O2 generation induced by Ca2+ is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.
Keywords: Mitochondria; Calcium; Oxidative stress; Mitochondrial permeability transition pore (mPTP); Reactive Oxygen Species; NADH;

The versatility of mitochondrial calcium signals: From stimulation of cell metabolism to induction of cell death by Alessandro Rimessi; Carlotta Giorgi; Paolo Pinton; Rosario Rizzuto (808-816).
Both the contribution of mitochondria to intracellular calcium (Ca2+) signalling and the role of mitochondrial Ca2+ uptake in shaping the cytoplasmic response and controlling mitochondrial function are areas of intense investigation. These studies rely on the appropriate use of emerging techniques coupled with judicious data interpretation to a large extent. The development of targeted probes based on the molecular engineering of luminescent proteins has allowed the specific measurement of Ca2+ concentration ([Ca2+]) and adenosine trisphosphate concentration ([ATP]) in intracellular organelles or cytoplasmic subdomains. This approach has given novel information on different aspects of mitochondrial homeostasis.

Novel mechanism of elimination of malfunctioning mitochondria (mitoptosis): Formation of mitoptotic bodies and extrusion of mitochondrial material from the cell by Konstantin G. Lyamzaev; Olga K. Nepryakhina; Valeria B. Saprunova; Lora E. Bakeeva; Olga Yu. Pletjushkina; Boris V. Chernyak; Vladimir P. Skulachev (817-825).
Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30–50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2–4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a “mitoptotic body”), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.
Keywords: Mitochondria; Mitoptosis; Mitoptotic body; Energy catastrophe; Reactive oxygen species;

Interrelated influence of superoxides and free fatty acids over mitochondrial uncoupling in skeletal muscle by Assunta Lombardi; Paola Grasso; Maria Moreno; Pieter de Lange; Elena Silvestri; Antonia Lanni; Fernando Goglia (826-833).
Mitochondrial uncoupling protein 3 (UCP3)-mediated uncoupling has been postulated to depend on several factors, including superoxides, free fatty acids (FFAs), and fatty acid hydroperoxides and/or their derivatives. We investigated whether there is an interrelation between endogenous mitochondrial superoxides and fatty acids in inducing skeletal muscle mitochondrial uncoupling, and we speculated on the possible involvement of UCP3 in this process. In the absence of FFAs, no differences in proton-leak kinetic were detected between succinate-energized mitochondria respiring in the absence or presence of rotenone, despite a large difference in complex I superoxide production. The addition of either arachidic acid or arachidonic acid induced an increase in proton-leak kinetic, with arachidonic acid having the more marked effect. The uncoupling effect of arachidic acid was independent of the presence of GDP, rotenone and vitamin E, while that of arachidonic acid was dependent on these factors. These data demonstrate that FFA and O2− play interrelated roles in inducing mitochondrial uncoupling, and we hypothesize that a likely formation of mitochondrial fatty acid hydroperoxides is a key event in the arachidonic acid-induced GDP-dependent inhibition of mitochondrial uncoupling.
Keywords: Mitochondria; Uncoupling protein 3; Proton leak;

Mitochondria in numerous cell types, especially in cultured cells, form a reticular network undergoing constant fusion and fission. The three dimensional (3D) morphology of these networks however has not been studied in detail to our knowledge. We have investigated insulinoma INS-1E and hepatocellular carcinoma HEP-G2 cells transfected with mitochondria-addressed GFP. Using 4Pi microscopy, 3D morphology changes responding to decreased oxidative phosphorylation and/or energetic status could be observed in these cells at an unprecedented 100 nm level of detail. In INS-1E cells cultivated at 11 mM glucose, the mitoreticulum appears predominantly as one interconnected mitochondrion with a nearly constant 262 ± 26 nm tubule diameter. If cultured at 5 mM glucose, INS-1E cells show 311 ± 36 nm tubules coexisting with numerous flat cisternae. Similar interconnected 284 ± 38 nm and 417 ± 110 nm tubules were found in HEP-G2 cells cultivated at 5 mM and hyperglycaemic 25 mM glucose, respectively. With rotenone inhibiting respiration to ~ 10%, disintegration into several reticula and numerous ~ 300 nm spheres or short tubules was observed. De-energization by uncoupling additionally led to formation of rings and bulky cisternae of 1.4 ± 0.4 μm diameter. Rotenone and uncoupler acted synergically in INS-1E cells and increased fusion (ongoing with fission) forming bowl-like shapes. In HEP-G2 cells fission partially ceased with FCCP plus rotenone. Thus we have revealed previously undescribed details for shapes upon mitochondrial disintegration and clearly demonstrate that high resolution 3D microscopy is required for visualization of mitochondrial network. We recommend 4Pi microscopy as a new standard.
Keywords: 3D morphology of mitochondrial network; 4Pi microscopy; Uncoupling; Oxidative phosphorylation; Insulinoma INS-1E cell; Hepatocellular carcinoma HEP-G2 cell;

In addition to their role in providing ATP for cellular functions via oxidative phosphorylation, mitochondria also play a critical role in initiating and/or regulating apoptosis through the release of proteins such as cytochrome c from intermembrane and intracristal compartments. The mechanism by which these proteins are able to cross the outer mitochondrial membrane has been a subject of controversy. This paper will review some recent results that demonstrate that inner mitochondrial membrane remodeling does occur during apoptosis in HeLa cells but does not appear to be a requirement for release of cytochrome c from intracristal compartments. Inner membrane remodeling does appear to be related to fragmentation of the mitochondrial matrix, and the form of the remodeling suggests a topological mechanism for inner membrane fission and fusion.
Keywords: Mitochondria; Apoptosis; Membrane remodeling; Electron microscopy; Electron tomography; Fluorescence microscopy;

Mitigation of NADH: Ubiquinone oxidoreductase deficiency by chronic Trolox treatment by Werner J.H. Koopman; Sjoerd Verkaart; Sjenet E. van Emst-de Vries; Sander Grefte; Jan A.M. Smeitink; Leo G.J. Nijtmans; Peter H.G.M. Willems (853-859).
Deficiency of mitochondrial NADH:ubiquinone oxidoreductase (complex I), is associated with a variety of clinical phenotypes such as Leigh syndrome, encephalomyopathy and cardiomyopathy. Circumstantial evidence suggests that increased reactive oxygen species (ROS) levels contribute to the pathogenesis of these disorders. Here we assessed the effect of the water-soluble vitamin E derivative Trolox on ROS levels, and the amount and activity of complex I in fibroblasts of six children with isolated complex I deficiency caused by a mutation in the NDUFS1, NDUFS2, NDUFS7, NDUFS8 or NDUFV1 gene. Patient cells displayed increased ROS levels and a variable decrease in complex I activity and amount. For control cells, the ratio between activity and amount was 1 whereas for the patients this ratio was below 1, indicating a defect in intrinsic catalytic activity of complex I in the latter cells. Trolox treatment dramatically reduced ROS levels in both control and patient cells, which was paralleled by a substantial increase in the amount of complex I. Although the ratio between the increase in activity and amount of complex I was exactly proportional in control cells it varied between 0.1 and 0.8 for the patients. Our findings suggest that the expression of complex I is regulated by ROS. Furthermore, they provide evidence that both the amount and intrinsic activity of complex I are decreased in inherited complex I deficiency. The finding that Trolox treatment increased the amount of complex I might aid the future development of antioxidant treatment strategies for patients. However, such treatment may only be beneficial to patients with a relatively small reduction in intrinsic catalytic defect of the complex.
Keywords: Mitochondria; Complex I deficiency; Reactive oxygen species; Vitamin E;

Damaged mitochondria can be eliminated in a process of organelle autophagy, termed mitophagy. In most cells, the organization of mitochondria in a network could interfere with the selective elimination of damaged ones. In principle, fission of this network should precede mitophagy; but it is unclear whether it is per se a trigger of autophagy. The pro-fission mitochondrial protein Fis1 induced mitochondrial fragmentation and enhanced the formation of autophagosomes which could enclose mitochondria. These changes correlated with mitochondrial dysfunction rather than with fragmentation, as substantiated by Fis1 mutants with different effects on organelle shape and function. In conclusion, fission associated with mitochondrial dysfunction stimulates an increase in autophagy.
Keywords: Mitochondria; Fission; Fis1; Autophagy;

A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase by Chris E. Cooper; Maria G. Mason; Peter Nicholls (867-876).
Nitric oxide can inhibit mitochondrial cytochrome oxidase in both oxygen competitive and uncompetitive modes. A previous model described these interactions assuming equilibrium binding to the reduced and oxidised enzyme respectively (Mason, et al. Proc. Natl. Acad. Sci. U S A 103 (2006) 708–713). Here we demonstrate that the equilibrium assumption is inappropriate as it requires unfeasibly high association constants for NO to the oxidised enzyme. Instead we develop a model which explicitly includes NO binding and its enzyme-bound conversion to nitrite. Removal of the nitrite complex requires electron transfer to the binuclear centre from haem a. This revised model fits the inhibition constants at any value of substrate concentration (ferrocytochrome c or oxygen). It predicts that the inhibited steady state should be a mixture of the reduced haem nitrosyl complex and the oxidized-nitrite complex. Unlike the previous model, binding to the oxidase is always proportional to the degree of inhibition of oxygen consumption. The model is consistent with data and models from a recent paper suggesting that the primary effect of NO binding to the oxidised enzyme is to convert NO to nitrite, rather than to inhibit enzyme activity (Antunes et al. Antioxid. Redox Signal. 9 (2007) 1569–1579).
Keywords: Nitrite; Enzyme kinetics; Nitric oxide; Mitochondria; Inhibition; Cytochrome oxidase;

Regulation of apoptosis by the redox state of cytochrome c by Guy C. Brown; Vilmante Borutaite (877-881).
Cytochrome c, released from mitochondria into the cytosol, triggers formation of the apoptosome resulting in activation of caspases. This paper reviews the evidence for and against the redox state of cytochrome c regulating apoptosis, and possible mechanisms of this. Three research groups have found that the oxidized form of cytochrome c (Fe3+) can induce caspase activation via the apoptosome, while the reduced form (Fe2+) cannot. It is unclear whether this is due to the oxidized and reduced forms of cytochrome c having: (i) different affinities for Apaf-1, (ii) different abilities to activate Apaf-1 once bound, or (iii) different affinities for other components of the cell. Experiments replacing the Fe of cytochrome c with redox-inactive metals indicate that cytochrome c does not have to change redox states to activate caspases. In healthy cells, cytosolic cytochrome c is rapidly reduced by various enzymes and/or reductants, which may function to block apoptosis. However, in apoptotic cells, cytosolic cytochrome c is rapidly oxidized by mitochondrial cytochrome oxidase, to which it has access due to permeabilization of the outer membrane. Regulation of the redox state of cytochrome c potentially enables regulation of the intrinsic pathway of apoptosis at a relatively late stage.
Keywords: Apoptosome; Cell death; Caspases; Cytochrome c; Cytochrome oxidase; Mitochondria;

Ischemic preconditioning (IPC) affords cardioprotection against ischemia–reperfusion (IR) injury, and while the molecular mechanisms of IPC are debated, the mitochondrial ATP-sensitive K+ channel (mKATP) has emerged as a candidate effector for several IPC signaling pathways. The molecular identity of this channel is unknown, but significant pharmacologic overlap exists between mKATP and mitochondrial respiratory complex II (succinate dehydrogenase). In this investigation, we utilized isolated cardiac mitochondria, Langendorff perfused hearts, and a variety of biochemical methods, to make the following observations: (i) The competitive complex II inhibitor malonate is formed in mitochondria under conditions resembling IPC. (ii) IPC leads to a reversible inhibition of complex II that has likely been missed in previous investigations due to the use of saturating concentrations of succinate. (iii) Malonate opens mKATP channels even when mitochondria are respiring on complex I-linked substrates, suggesting an effect of this inhibitor on the mKATP channel independent of complex II inhibition. Together, these observations suggest that complex II inhibition by endogenously formed malonate may represent an important activation pathway for mKATP channels during IPC.
Keywords: ATP-sensitive potassium channel; mKATP; Preconditioning; Ischemia; Succinate dehydrogenase; Diazoxide; Mitochondria;

Prevention of leak in the proton pump of cytochrome c oxidase by Ville R.I. Kaila; Michael Verkhovsky; Gerhard Hummer; Mårten Wikström (890-892).
The cytochrome c oxidases (CcO), which are responsible for most O2 consumption in biology, are also redox-linked proton pumps that effectively convert the free energy of O2 reduction to an electrochemical proton gradient across mitochondrial and bacterial membranes. Recently, time-resolved measurements have elucidated the sequence of events in proton translocation, and shed light on the underlying molecular mechanisms. One crucial property of the proton pump mechanism has received less attention, viz. how proton leaks are avoided. Here, we will analyse this topic and demonstrate how the key proton-carrying residue Glu-242 (numbering according to the sequence of subunit I of bovine heart CcO) functions as a valve that has the effect of minimising back-leakage of the pumped proton.
Keywords: Oxygen reductionGlutamic acid">Proton transfer; Oxygen reduction; Glutamic acid;

We have studied the effects of rotenone in myoblasts from healthy donors and from patients with Ullrich congenital muscular dystrophy (UCMD), a severe muscle disease due to mutations in the genes encoding the extracellular matrix protein collagen VI. Addition of rotenone to normal myoblasts caused a very limited mitochondrial depolarization because the membrane potential was maintained by the F1FO synthase, as indicated by full depolarization following the subsequent addition of oligomycin. In UCMD myoblasts rotenone instead caused complete mitochondrial depolarization, which was followed by faster ATP depletion than in healthy myoblasts. Mitochondrial depolarization could be prevented by treatment with cyclosporin A and intracellular Ca2+ chelators, while it was worsened by depleting Ca2+ stores with thapsigargin. Thus, in UCMD myoblasts rotenone-induced depolarization is due to opening of the permeability transition pore rather than to inhibition of electron flux as such. These findings indicate that in UCMD myoblasts the threshold for pore opening is very close to the resting membrane potential, so that even a small depolarization causes permeability transition pore opening and precipitates ATP depletion.
Keywords: Mitochondria; Permeability transition; Cyclosporin A; Collagen VI; Muscular dystrophy;

Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway by Håkan Lepp; Lina Salomonsson; Jia-Peng Zhu; Robert B. Gennis; Peter Brzezinski (897-903).
Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O2 to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O2 reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O2 reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK a of E286, an internal proton donor to the catalytic site, from 9.4 to ~ 11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O2 reduction, but a decrease in the apparent pK a of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.
Keywords: Proton transfer; Electron transfer; Heme-copper oxidase; Membrane protein; Proton pump; Redox;

Biogenesis of cytochrome c oxidase — in vitro approaches to study cofactor insertion into a bacterial subunit I by Peter Greiner; Achim Hannappel; Carolin Werner; Bernd Ludwig (904-911).
Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of cytochrome c oxidase using the soil bacterium Paracoccus denitrificans as a model organism. The use of bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is involved in copper delivery to the CuB center, has been purified and characterized spectroscopically. A previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of “solubilized” subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes.
Keywords: Assembly; Cox11; Copper chaperone; Heme a; Cell-free expression; Paracoccus denitrificans;

Carboxyl groups of possible functional importance in bovine and bacterial cytochrome c oxidases (CcO) are reviewed and assessed. A critical analysis is presented of available mid-infrared vibrational data that pertain to these functional carboxyl groups. These data and their interpretations are discussed in relation to current models of the mechanism of proton and electron coupling in the protonmotive CcO superfamily.
Keywords: Cytochrome c oxidase; FTIR spectroscopy; Infrared spectroscopy; Proton transfer; Energy coupling;

Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase by Sofia M. Kapetanaki; Sarah J. Field; Ross J.L. Hughes; Nicholas J. Watmough; Ursula Liebl; Marten H. Vos (919-924).
The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in ~ 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site.
Keywords: Nitric oxide reductase; Ligand dynamics; Ultrafast spectroscopy; Heme; Dinuclear center;

The catalytic mechanism of heme–copper oxidases – electron transfer coupled to proton pumping – is not yet fully understood. Single turnover experiments in which fully reduced cytochrome aa 3 from Paracoccus denitrificans reacts with O2 using the microsecond freeze-hyperquenching sampling technique enabled trapping of transient catalytic intermediates and analysis by low temperature UV–Visible, X-band and Q-band EPR spectroscopy. Our recent findings (Wiertz et al. (2007) J. Biol. Chem. 282, 31580–31591), which show that the strictly conserved W272 is a redox active residue are reviewed here. The W272 forms a tryptophan neutral radical in the transition F → FW⁎ → OH in which the novel intermediate FW⁎ harbors the tryptophan radical. The potential role of W272 in proton pumping is highlighted.
Keywords: Cytochrome c oxidase; Proton pumping; Tryptophan; Pre-steady state kinetics; Electron Paramagnetic Resonance;

Looking for the minimum common denominator in haem–copper oxygen reductases: Towards a unified catalytic mechanism by Manuela M. Pereira; Filipa L. Sousa; Andreia F. Veríssimo; Miguel Teixeira (929-934).
Haem–copper oxygen reductases are transmembrane protein complexes that reduce dioxygen to water and pump protons across the mitochondrial or periplasmatic membrane, contributing to the transmembrane difference of electrochemical potential. Seven years ago we proposed a classification of these enzymes into three different families (A, B and C), based on the amino acid residues of their proton channels and amino acid sequence comparison, later supported by the so far identified characteristics of the catalytic centre of members from each family. The three families have in common the same general structural fold of the catalytic subunit, which contains the same or analogous prosthetic groups, and proton channels. These observations raise the hypothesis that the mechanisms for dioxygen reduction, proton pumping and the coupling of the two processes may be the same for all these enzymes. Under this hypothesis, they should be performed and controlled by the same or equivalent elements/events, and the identification of retained elements in all families will reveal their importance and may prompt the definition of the enzyme operating mode. Thus, we believe that the search for a minimum common denominator has a crucial importance, and in this article we highlight what is already established for the haem–copper oxygen reductases and emphasize the main questions still unanswered in a comprehensive basis.
Keywords: Haem–copper oxygen reductase; Cytochrome oxidase; Quinol oxidase; Electron–proton coupling;

Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells by Malou Friederich; Angelica Fasching; Peter Hansell; Lina Nordquist; Fredrik Palm (935-940).
We have previously reported increased O2 consumption unrelated to active transport by tubular cells and up-regulated mitochondrial uncoupling protein (UCP)-2 expressions in diabetic kidneys. It is presently unknown if the increased UCP-2 levels in the diabetic kidney results in mitochondrial uncoupling and increased O2 consumption, which we therefore investigated in this study. The presence of UCP-2 in proximal tubular cells was confirmed by immunohistochemistry and found to be increased (western blot) in homogenized tissue and isolated mitochondria from kidney cortex of diabetic rats. Isolated proximal tubular cells had increased total and ouabain-insensitive O2 consumption compared to controls. Isolated mitochondria from diabetic animals displayed increased glutamate-stimulated O2 consumption (in the absence of ADP and during inhibition of the ATP-synthase by oligomycin) compared to controls. Guanosine diphosphate, an UCP inhibitor, and bovine serum albumin which removes fatty acids that are essential for UCP-2 uncoupling activity, independently prevented the increased glutamate-stimulated O2 consumption in mitochondria from diabetic animals. In conclusion, diabetic rats have increased mitochondrial UCP-2 expression in renal proximal tubular cells, which results in mitochondrial uncoupling and increased O2 consumption. This mechanism may be protective against diabetes-induced oxidative stress, but will increase O2 usage. The subsequently reduced O2 availability may contribute to diabetes-induced progressive kidney damage.
Keywords: Diabetes mellitus; Kidney; Mitochondria; Uncoupling protein-2; Oxygen consumption;

The study of the pathogenic mechanism of mitochondrial diseases provides information on basic bioenergetics by Giancarlo Solaini; David A. Harris; Giorgio Lenaz; Gianluca Sgarbi; Alessandra Baracca (941-945).
Mitochondrial F1F0-ATPase was studied in lymphocytes from patients with neuropathy, ataxia, and retinitis pigmentosa (NARP), caused by a mutation at leu-156 in the ATPase 6 subunit. The mutation giving the milder phenotype (Leu156Pro) suffered a 30% reduction in proton flux, and a similar loss in ATP synthetic activity. The more severe mutation (Leu156Arg) also suffered a 30% reduction in proton flux, but ATP synthesis was virtually abolished. Oligomycin sensitivity of the proton translocation through F0 was enhanced by both mutations. We conclude that in the Leu156Pro mutation, rotation of the c-ring is slowed but coupling of ATP synthesis to proton flux is maintained, whereas in the Leu156Arg mutation, proton flux appears to be uncoupled. Modelling indicated that, in the Leu156Arg mutation, transmembrane helix III of ATPase 6 is unable to span the membrane, terminating in an intramembrane helix II–helix III loop. We propose that the integrity of transmembrane helix III is essential for the mechanical function of ATPase 6 as a stator element in the ATP synthase, but that it is not relevant for oligomycin inhibition.
Keywords: Mitochondria; ATP synthase; ATPase 6 subunit; Proton transport; mtDNA mutation;

The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, especially necrosis, and mediates the injury tissues such as the heart and brain experience following ischaemia and reperfusion. However, the molecular identity of the MPTP remains uncertain. Knockout studies have confirmed a role for cyclophilin-D (CyP-D) in pore opening, probably mediated by its peptidyl–prolyl cistrans isomerase activity that facilitates a conformational change in an inner membrane protein. However, similar knockout studies have cast doubt on the central role of the adenine nucleotide translocase (ANT), previously regarded as a leading contender for the membrane component that forms the transmembrane channel of the MPTP. Here we review the evidence for and against a role for the ANT in MPTP opening and conclude that it usually plays a regulatory role rather than provide the transmembrane pore component. We suggest that the protein fulfilling the latter role is the mitochondrial phosphate carrier (PiC) and summarise recent evidence in support of this proposal. Our data are consistent with a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. We propose that this is enhanced by an association of the PiC with the “c” conformation of the ANT. Agents that modulate pore opening may act on either or both the PiC and the ANT.
Keywords: Adenine nucleotide translocase; Cyclophilin-D; Mitochondrial phosphate carrier; Permeability transition; Ischaemia; Reperfusion; Oxidative stress; Calcium;

Glutamate excitotoxicity amplifies neuronal death following stroke. We have explored the mechanisms underlying the collapse of mitochondrial potential (Δψ m) and loss of [Ca2+]c homeostasis in rat hippocampal neurons in culture following toxic glutamate exposure. The collapse of Δψ m is multiphasic and Ca2+-dependent. Glutamate induced a decrease in NADH autofluorescence which preceded the loss of Δψ m. Both the decrease in NADH signal and the loss of Δψ m were suppressed by Ru360 and both were delayed by inhibition of PARP (by 3-AB or DPQ). During this period, addition of mitochondrial substrates (methyl succinate and TMPD–ascorbate) or buffering [Ca2+]i (using BAPTA-AM or EGTA-AM), rescued Δψ m. These data suggest that mitochondrial Ca2+ uptake activates PARP which in turn depletes NADH, promoting the initial collapse of Δψ m. After > ~ 20 min, buffering Ca2+ or substrate addition failed to restore Δψ m. In neurons from cyclophilin D−/− (cypD−/−) mice or in cells treated with cyclosporine A, removal of Ca2+ restored Δψ m even after 20 min of glutamate exposure, suggesting involvement of the mPTP in the irreversible depolarisation seen in WT cells. Thus, mitochondrial depolarisation represents two consecutive but distinct processes driving cell death, the first of which is reversible while the second is not.
Keywords: Mitochondria; Neuron; Glutamate; Excitotoxicity; Intracellular calcium;

Tissue specificity of mitochondrial glutamate pathways and the control of metabolic homeostasis by Francesca Frigerio; Marina Casimir; Stefania Carobbio; Pierre Maechler (965-972).
Glutamate is implicated in numerous metabolic and signalling functions that vary according to specific tissues. Glutamate metabolism is tightly controlled by activities of mitochondrial enzymes and transmembrane carriers, in particular glutamate dehydrogenase and mitochondrial glutamate carriers that have been identified in recent years. It is remarkable that, although glutamate-specific enzymes and transporters share similar properties in most tissues, their regulation varies greatly according to particular organs in order to achieve tissue specific functions. This is illustrated in this review when comparing glutamate handling in liver, brain, and pancreatic β-cells. We describe the main cellular glutamate pathways and their specific functions in different tissues, ultimately contributing to the control of metabolic homeostasis at the organism level.
Keywords: Glutamate; Mitochondria; Glutamate dehydrogenase; Glutamate carrier; Pancreatic beta-cell; SIRT;

On the role of uncoupling protein-2 in pancreatic beta cells by Charles Affourtit; Martin D. Brand (973-979).
Pancreatic beta cells secrete insulin when blood glucose levels are high. Dysfunction of this glucose-stimulated insulin secretion (GSIS) is partly responsible for the manifestation of type 2 diabetes, a metabolic disorder that is rapidly becoming a global pandemic. Mitochondria play a central role in GSIS by coupling glucose oxidation to production of ATP, a signal that triggers a series of events that ultimately leads to insulin release. Beta cells express a mitochondrial uncoupling protein, UCP2, which is rather surprising as activity of such a protein is anticipated to lower the efficiency of oxidative phosphorylation, and hence to impair GSIS. The mounting evidence demonstrating that insulin secretion is indeed blunted by UCP2 agrees with this prediction, and has provoked the idea that UCP2 activity contributes to beta cell pathogenesis and development of type 2 diabetes. Although this notion may be correct, the evolved function of UCP2 remains unclear. With this paper we aim to provide a brief account of the present state of affairs in this field, suggest a physiological role for UCP2, and highlight some of our own recent results.
Keywords: Pancreatic beta cells; Uncoupling protein-2; Mitochondrial respiration; Coupling efficiency; Glucose-stimulated insulin secretion; Type 2 diabetes;

Cytochrome c assembly: A tale of ever increasing variation and mystery? by Stuart J. Ferguson; Julie M. Stevens; James W.A. Allen; Ian B. Robertson (980-984).
Formation of cytochromes c requires a deceptively simple post-translational modification, the formation of two thioether bonds (or rarely one) between the thiol groups of two cysteine residues found in a CXXCH motif (with some occasional variations) and the vinyl groups of heme. There are three partially characterised systems for facilitating this post-translational modification; within these systems there is also variation. In addition, there are clear indications for two other distinct systems. Here some of the current issues in understanding the systems are analysed.
Keywords: c-type cytochrome; Periplasm; Post-translational modification; Heme; Thioether bond formation; Ccm system;

The past and present of sodium energetics: May the sodium-motive force be with you by Armen Y. Mulkidjanian; Pavel Dibrov; Michael Y. Galperin (985-992).
All living cells routinely expel Na+ ions, maintaining lower concentration of Na+ in the cytoplasm than in the surrounding milieu. In the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of Na+ occurs at the expense of the proton-motive force. Some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of Na+ (sodium-motive force). These primary Na+ pumps have been traditionally seen as adaptations to high external pH or to high temperature. Subsequent studies revealed, however, the mechanisms for primary sodium pumping in a variety of non-extremophiles, such as marine bacteria and certain bacterial pathogens. Further, many alkaliphiles and hyperthermophiles were shown to rely on H+, not Na+, as the coupling ion. We review here the recent progress in understanding the role of sodium-motive force, including (i) the conclusion on evolutionary primacy of the sodium-motive force as energy intermediate, (ii) the mechanisms, evolutionary advantages and limitations of switching from Na+ to H+ as the coupling ion, and (iii) the possible reasons why certain pathogenic bacteria still rely on the sodium-motive force.
Keywords: Sodium energetics; Sodium-motive force; ATP synthase; Nanomotors; Proton transfer; Sodium-dependent transporters; Biological membranes; Vibrio cholerae; Chlamydia sp.; Anaerobic pathogenic bacteria;

Regulation of thermogenesis in flowering Araceae: The role of the alternative oxidase by Anneke M. Wagner; Klaas Krab; Marijke J. Wagner; Anthony L. Moore (993-1000).
The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.
Keywords: Thermoregulation; Plant respiration; Alternative oxidase; Ubiquinone; Flux control analysis;

The Q-cycle reviewed: How well does a monomeric mechanism of the bc 1 complex account for the function of a dimeric complex? by Antony R. Crofts; J. Todd Holland; Doreen Victoria; Derrick R.J. Kolling; Sergei A. Dikanov; Ryan Gilbreth; Sangmoon Lhee; Richard Kuras; Mariana Guergova Kuras (1001-1019).
Recent progress in understanding the Q-cycle mechanism of the bc 1 complex is reviewed. The data strongly support a mechanism in which the Qo-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Qo-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c 1 and liberate the H+. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O2 is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b L to enhance the rate constant. The acceptor reactions at the Qi-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b 150 phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc 1 complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.
Keywords: Q-cycle; Constraints on molecular mechanism; bc 1 complex; Kinetic model; Coulombic interaction; Thermodynamic model;

Cardiolipin as an oxidative target in cardiac mitochondria in the aged rat by Edward J. Lesnefsky; Charles L. Hoppel (1020-1027).
The aged heart sustains greater injury during ischemia (ISC) and reperfusion (REP) compared to the adult heart. In the Fischer 344 (F344) rat, aging decreases oxidative phosphorylation and complex III activity increasing the production of reactive oxygen species in interfibrillar mitochondria (IFM) located among the myofibrils. In the isolated, perfused 24 month old elderly F344 rat heart 25 min of stop–flow ISC causes additional damage to complex III, further decreasing the rate of oxidative phosphorylation. We did not observe further progressive mitochondrial damage during REP. We next asked if ISC or REP increased oxidative damage within mitochondria of the aged heart. Cardiolipin (CL) is a phospholipid unique to mitochondria consisting predominantly of four linoleic acid residues (C18:2). Following ISC and REP in the aged heart, there is a new CL species containing three oxygen atoms added to one linoleic residue. ISC alone was sufficient to generate this new oxidized molecular species of CL. Based upon oxidative damage to CL, complex III activity, and oxidative phosphorylation, mitochondrial damage thus occurs in the aged heart mainly during ISC, rather than during REP. Mitochondrial damage during ischemia sets the stage for mitochondrial-driven cardiomyocyte injury during reperfusion in the aged heart.
Keywords: Ischemia; Reperfusion; Heart; Ubiquinone:cytochrome c reductase (complex III); Cardiolipin; Mitochondria; Aging;

Targeting lipophilic cations to mitochondria by Michael P. Murphy (1028-1031).
Mitochondrial function and dysfunction contributes to a range of important aspects of biomedical research. Consequently there is considerable interest in developing approaches to modify and report on mitochondria in cells and in vivo. One approach has been to target bioactive molecules to mitochondria by conjugating them to lipophilic cations. Due to the large mitochondrial membrane potential, the cations are accumulated within mitochondria inside cells. This approach had been used to develop mitochondria-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death and also to develop probes of mitochondrial function. Here we outline some of the background to the development of these compounds.
Keywords: Mitochondria; Antioxidant; MitoQ; Oxidative damage;

Distance metrics for heme protein electron tunneling by Christopher C. Moser; Sarah E. Chobot; Christopher C. Page; P. Leslie Dutton (1032-1037).
There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 Å longer. In a typical protein medium, such a distance difference normally corresponds to a ~ 1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.
Keywords: Protein electron tunneling; Heme; Ruthenium; Electron transfer;

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc 1 complex in the past have led to the formulation of the “protonmotive Q-cycle” mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the QP site with both electrons transferred simultaneously to ISP and cyt b L when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc 1 demonstrates that the reduced ISP-ED moves to the c 1-position to reduce cyt c 1 only after the reduced cyt b L is oxidized by cyt b H. However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of QP site inhibitors, Pm and Pf, under various redox states of the bc 1 complex, suggest that the electron transfer from heme b L to b H is the driving force for the releasing of the reduced ISP-ED from the b-position to c 1-position to reduce cyt c 1.
Keywords: Cytochrome; Electron transfer; Iron–sulfur protein; Inhibitor;

Energy transduction in the cytochrome bc 1 complex is achieved by catalyzing opposite oxido-reduction reactions at two different quinone binding sites. We have determined the pre-steady state kinetics of cytochrome b and c 1 reduction at varying quinol/quinone ratios in the isolated yeast bc 1 complex to investigate the mechanisms that minimize inhibition of quinol oxidation at center P by reduction of the b H heme through center N. The faster rate of initial cytochrome b reduction as well as its lower sensitivity to quinone concentrations with respect to cytochrome c 1 reduction indicated that the b H hemes equilibrated with the quinone pool through center N before significant catalysis at center P occurred. The extent of this initial cytochrome b reduction corresponded to a level of b H heme reduction of 33%–55% depending on the quinol/quinone ratio. The extent of initial cytochrome c 1 reduction remained constant as long as the fast electron equilibration through center N reduced no more than 50% of the b H hemes. Using kinetic modeling, the resilience of center P catalysis to inhibition caused by partial pre-reduction of the b H hemes was explained using kinetics in terms of the dimeric structure of the bc 1 complex which allows electrons to equilibrate between monomers.
Keywords: bc 1 complex; Electron transfer; Quinone; Semiquinone;

The loneliness of the electrons in the bc 1 complex by Stéphane Ransac; Nicolas Parisey; Jean-Pierre Mazat (1053-1059).
A stochastic approach based on Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between individual unique redox centres of a given complex and not populations of electrons and redox centres as usually considered in ordinary differential equations. In this way we approach the molecular functioning of the bc 1 complex based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equation. The main features of our simulations are the dominant and robust emergence of a Q-cycle mechanism and the near absence of short-circuits in the normal functioning of the bc 1 complex. Thus, in our paper, the Mitchell Q-cycle no longer appears as an a priori hypothesis but arises out of the bc 1 complex structure and of the kinetic laws of redox reactions.
Keywords: Gillespie algorithm; bc 1 complex; Stochastic modelling; Q-cycle; Short-circuit;

Identification of serine phosphorylation in mitochondrial uncoupling protein 1 by Audrey M. Carroll; Richard K. Porter; Nick A. Morrice (1060-1065).
Native uncoupling protein 1 was purified from rat brown adipose tissue of cold-acclimated rats and rats kept at room temperature, in the presence of phosphatase inhibitors. The purified protein from cold-acclimated animals was digested with trypsin and immobilized metal affinity chromatography was used to select for phosphopeptides. Tandem mass spectroscopic analysis of the peptides derived from uncoupling protein 1, suggests phosphorylation of serine 3 or 4 and identified phosphorylation of serine 51. Furthermore, we were able to demonstrate that antibodies to phosphoserine detect full-length UCP 1 and that the proportion of phosphoserine on UCP1, purified from cold-acclimated rats, was significantly greater than that on UCP 1 from rats kept at room temperature (90 ± 4% compared to 62 ± 8%, p  = 0.013), respectively). We conclude that uncoupling protein 1 is a phosphoprotein and that cold-acclimation increases the proportion of UCP1 that is serine phosphorylated.
Keywords: Uncoupling protein-1; Brown adipose tissue; Covalent modification; Serine phosphorylation; Tandem mass spectroscopy;

Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration by Hong Yu; Icksoo Lee; Arthur R. Salomon; Kebing Yu; Maik Hüttemann (1066-1071).
Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc 1 complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s− 1 whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s− 1. Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.
Keywords: Mitochondria; Oxidative phosphorylation; Cell signaling; Cytochrome c; Cytochrome c oxidase; Apoptosis; Respiration;

Mitochondrial comparative proteomics: Strengths and pitfalls by Grégory Mathy; Francis E. Sluse (1072-1077).
In this review, we describe the various techniques available to carry out valid comparative proteomics, their advantages and their disadvantages according to the goal of the research. Two-dimensional electrophoresis and 2D-DIGE are compared to shotgun proteomics and SILE. We give our opinion on the best fields of application in the domain of comparative proteomics. We emphasize the usefulness of these new tools, providing mass data to study physiology and mitochondrial plasticity when faced with a specific mitochondrial insufficiency or exogenic stress. We illustrate the subject with results obtained in our laboratory specifying the importance of an approach of comparative proteomics combined from mitochondria and from the cell, which makes it possible to obtain important information on the status of the mitochondrial function at the cellular level. Finally, we draw attention to the dangers of the extrapolation of proteomic data to metabolic flows which requires the greatest care.
Keywords: Mitochondria; Proteomics; Mass spectrometry;