BBA - Bioenergetics (v.1658, #1-2)

Preface by B. Andrea Melandri; Giancarlo Solaini (1).

NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter by E Padan; T Tzubery; K Herz; L Kozachkov; A Rimon; L Galili (2-13).
Na+/H+ antiporters are ubiquitous membrane proteins that are involved in homeostasis of H+ and Na+ throughout the biological kingdom. Corroborating their role in pH homeostasis, many of the Na+/H+ antiporter proteins are regulated directly by pH. The pH regulation of NhaA, the Escherichia coli Na+/H+ antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na+/H+ antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity. Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules. Importantly, in the NhaA family, these domains are conserved. Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain. Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved. Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.
Keywords: Membrane protein; Active transport; Na+/H+ antiporter; NhaA; pH-regulation;

In the last few years, three laboratories have reported three entirely different crystallographic models for the L photointermediate of bacteriorhodopsin. All are from X-ray diffraction of illuminated crystals that contain L in photostationary states created at similar cryogenic temperatures. This article compares the models and their implications, the crystallographic statistics and the methods used to derive them, as well as their agreement with non-crystallographic information.
Keywords: Bacteriorhodopsin; Membrane protein; Crystallography; Retinal; Photointermediate;

Cooperative effects are fundamental for electroprotonic energy transduction processes, crucial to sustain much of life chemistry. However, the primary cooperative mechanism by which transmembrane proteins couple the downhill transfer of electrons to the uphill activation (acidification) of protic groups is still a matter of great controversy.To understand cooperative processes fully, it is necessary to obtain the microscopic thermodynamic parameters of the functional centres and relate them to the relevant structural features, a task difficult to achieve for large proteins. The approach discussed here explores how this may be done by extrapolation from mechanisms used by simpler proteins operative in similar processes.The detailed study of small, soluble cytochromes performing electroprotonic activation has shown how they use anti-electrostatic effects to control the synchronous movement of charges. These include negative e/H+ (redox-Bohr effect) cooperativities. This capacity is the basis to discuss an unorthodox mechanism consistent with the available experimental data on the process of electroprotonic energy transduction performed by cytochrome c oxidase (CcO).
Keywords: Cooperativity; Anti-Coulomb processes; Cytochrome c 3; Synchronization;

In order to circumvent deleterious effects of hypo- and hyperosmotic conditions in its environment, Corynebacterium glutamicum has developed a number of mechanisms to counteract osmotic stress. The first response to an osmotic upshift is the activation of uptake mechanisms for the compatible solutes betaine, proline, or ectoine, namely BetP, EctP, ProP, LcoP and PutP. BetP, the most important uptake system responds to osmotic stress by regulation at the level of both protein activity and gene expression. BetP was shown to harbor three different properties, i.e. catalytic activity (betaine transport), sensing of appropriate stimuli (osmosensing) and signal transduction to the catalytic part of the carrier protein which adapts its activity to the extent of osmotic stress (osmoregulation). BetP is comprised of 12 transmembrane segments and carries N- and C-terminal domains, which are involved in osmosensing and/or osmoregulation. Recent results on molecular properties of these domains indicate the significance of particular amino acids within the terminal 25 amino acids of the C-terminal domain of BetP for the process of osmosensing and osmoregulation.
Keywords: Corynebacterium glutamicum; Solute transport; Osmostress; Osmoregulation; Osmosensing; Betaine;

The process of mitochondrial protein import has been studied for many years. Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood. Insight into one of these processes, assembly of β-barrel proteins into the mitochondrial outer membrane, will be discussed. This review focuses on recent data that suggest that assembly of β-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex). Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of β-barrel assembly into membranes has been conserved through evolution.
Keywords: Mitochondria; Protein sorting; TOM complex; SAM complex; Porin; Protein assembly;

NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.
Keywords: Nitric oxide; Mitochondria; Complex I; NADH-ubiquinone oxidoreductase; Parkinson's disease; Respiration;

Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose–water amorphous matrices by Francesco Francia; Gerardo Palazzo; Antonia Mallardi; Lorenzo Cordone; Giovanni Venturoli (50-57).
The coupling between electron transfer and protein dynamics has been studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides by embedding the protein into room temperature solid trehalose–water matrices. Electron transfer kinetics from the primary quinone acceptor (QA ) to the photoxidized donor (P+) were measured as a function of the duration of photoexcitation from 20 ns (laser flash) to more than 1 min. Decreasing the water content of the matrix down to ≈5×103 water molecules per RC causes a reversible four-times acceleration of P+QA recombination after the laser pulse. By comparing the broadly distributed kinetics observed under these conditions with the ones measured in glycerol–water mixtures at cryogenic temperatures, we conclude that RC relaxation from the dark-adapted to the light-adapted state and thermal fluctuations among conformational substates are hindered in the room temperature matrix over the time scale of tens of milliseconds. When the duration of photoexcitation is increased from a few milliseconds to the second time scale, recombination kinetics of P+QA slows down progressively and becomes less distributed, indicating that even in the driest matrices, during continuous illumination, the RC is gaining a limited conformational freedom that results in partial stabilization of P+QA . This behavior is consistent with a tight structural and dynamical coupling between the protein surface and the trehalose–water matrix.
Keywords: Photosynthetic reaction center; Trehalose; Electron transfer; Protein dynamics; Conformational relaxation;

The alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) alters DNA and stimulates the activity of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair. The consumption of cellular NAD+ by PARP-1 is accompanied by ATP depletion, mitochondrial depolarization and release of proapoptotic proteins, but whether a causal relationship exists among these events remains an open question. Most of cellular NAD+ is stored in the mitochondrial matrix and becomes available for cytosolic and nuclear processes only after its release through the permeability transition pore (PTP), a voltage-gated inner membrane channel. Here we have explored whether MNNG affects mitochondrial function upstream of PARP-1 activation. We show that MNNG has a dual effect on isolated mitochondria. At relatively low concentrations (up to 0.1 mM), it selectively sensitizes the PTP to opening, while at higher concentrations (above 0.5 mM) it inhibits carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-stimulated respiration. MNNG caused PTP opening and activation of the mitochondrial proapoptotic pathway in intact HeLa cells, which resulted in cell death that could be prevented by the PTP inhibitor CsA. We conclude that a key event in MNNG-dependent cell death is induction of PTP opening that occurs independently of PARP-1 activation.
Keywords: NAD+; Apoptosis; PARP; Permeability transition;

Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle by Nathaniel Traaseth; Sarah Elfering; Joseph Solien; Virginia Haynes; Cecilia Giulivi (64-71).
An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the presence of either l-Arg (to sustain a mtNOS activity) or N G -monomethyl-l-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K 0.5 of 0.16 μM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with l-Arg and with NMMA at various calcium concentrations, a K 0.5 of 1.2 μM intramitochondrial free calcium was obtained, similar to the K 0.5 (0.9 μM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion.
Keywords: Mitochondria; Nitric oxide; Calcium; Krebs cycle; Dehydrogenase; Cytochrome oxidase; Oxygen; Nitric oxide synthase;

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O–H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D2O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O–D stretch at 2171 cm−1 as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the “hydration switch model”, hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK a of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.
Keywords: Proton pump; Retinal; Hydrogen bond; Fourier transform infrared spectroscopy; Internal water molecule; Isotope effect;

Mitochondrial diseases by Salvatore DiMauro (80-88).
By convention, the term “mitochondrial diseases” refers to disorders of the mitochondrial respiratory chain, which is the only metabolic pathway in the cell that is under the dual control of the mitochondrial genome (mtDNA) and the nuclear genome (nDNA). Therefore, a genetic classification of the mitochondrial diseases distinguishes disorders due to mutations in mtDNA, which are governed by the relatively lax rules of mitochondrial genetics, and disorders due to mutations in nDNA, which are governed by the stricter rules of mendelian genetics.Mutations in mtDNA can be divided into those that impair mitochondrial protein synthesis in toto and those that affect any one of the 13 respiratory chain subunits encoded by mtDNA. Essential clinical features for each group of diseases are reviewed.Disorders due to mutations in nDNA are more abundant not only because most respiratory chain subunits are nucleus-encoded but also because correct assembly and functioning of the respiratory chain require numerous steps, all of which are under the control of nDNA. These steps (and related diseases) include: (i) synthesis of assembly proteins; (ii) intergenomic signaling; (iii) mitochondrial importation of nDNA-encoded proteins; (iv) synthesis of inner mitochondrial membrane phospholipids; (v) mitochondrial motility and fission.
Keywords: Mitochondrial disease; Respiratory chain; mtDNA mutation; nDNA mutation;

Bioenergetics of mitochondrial diseases associated with mtDNA mutations by Giorgio Lenaz; Alessandra Baracca; Valerio Carelli; Marilena D'Aurelio; Gianluca Sgarbi; Giancarlo Solaini (89-94).
This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure.Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex.Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis.A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown.In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.
Keywords: Mitochondrial cytopathy; LHON; NARP; Complex I; ATP synthase; Cytochrome oxidase subunit III;

Protonmotive cooperativity in cytochrome c oxidase by Sergio Papa; Nazzareno Capitanio; Giuseppe Capitanio; Luigi L Palese (95-105).
Cooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins. Analysis of proton/electron coupling at the four redox centers, i.e. CuA, heme a, heme a 3 and CuB, in the purified bovine cytochrome c oxidase in the unliganded, CO-liganded and CN-liganded states is presented. These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers.Heme a (and CuA) exhibits a cooperative proton/electron linkage (Bohr effect). Bohr effect seems also to be associated with the oxygen-reduction chemistry at the heme a 3–CuB binuclear center. Data on electron transfer in cytochrome c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from CuA to the binuclear center in addition to electron transfer via heme a.A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme a in the proton pump of cytochrome c oxidase is presented. On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined.
Keywords: Cytochrome c oxidase; Proton pumping; Cooperativity;

The vacuolar-type H+-ATPases (V-ATPase) are a family of multi-subunit ATP-dependent proton pumps involved in a wide variety of physiological processes. They are present in endomembrane organelles such as vacuoles, lysosomes, endosomes, the Golgi apparatus, chromaffin granules and coated vesicles, and acidify the luminal pH of these intracellular compartments. They also pump protons across the plasma membranes of specialized cells including osteoclasts and epithelial cells in kidneys and male genital tracts. Here, we briefly summarize our recent studies on the diverse and essential roles of mammalian V-ATPase.
Keywords: V-ATPase; Endomembrane organelle; Vesicle transport; Subunit isoform; Luminal acidification;

Mitochondrial diseases and ATPase defects of nuclear origin by Josef Houštěk; Tomáš Mráček; Alena Vojtı́šková; Jiřı́ Zeman (115-121).
Dysfunctions of the F1Fo-ATPase complex cause severe mitochondrial diseases affecting primarily the paediatric population. While in the maternally inherited ATPase defects due to mtDNA mutations in the ATP6 gene the enzyme is structurally and functionally modified, in ATPase defects of nuclear origin mitochondria contain a decreased amount of otherwise normal enzyme. In this case biosynthesis of ATPase is down-regulated due to a block at the early stage of enzyme assembly—formation of the F1 catalytic part. The pathogenetic mechanism implicates dysfunction of Atp12 or other F1-specific assembly factors. For cellular energetics, however, the negative consequences may be quite similar irrespective of whether the ATPase dysfunction is of mitochondrial or nuclear origin.
Keywords: Mitochondrial disease; Cardiomyopathy; ATP synthase; Oxidative phosphorylation; Respiratory chain complex;

Understanding aging: revealing order out of chaos by Eric Dufour; Nils-Göran Larsson (122-132).
Aging is often described as an extremely complex process affecting all of the vital parameters of an individual. In this article, we review how understanding of aging evolved from the first analyses of population survival to the identification of the molecular mechanisms regulating life span. Abundant evidence implicates mitochondria in aging and we focus on the three main components of the mitochondrial theory of aging: (1) increased reactive oxygen species (ROS) production, (2) mitochondrial DNA (mtDNA) damage accumulation, and (3) progressive respiratory chain dysfunction. Experimental evidence shows a relationship between respiratory chain dysfunction, ROS damage, and aging in most of the model organisms. However, involvement of the mtDNA mutations in the aging process is still debated. We recently created a mutant mouse strain with increased levels of somatic mtDNA mutations causing a progressive respiratory chain deficiency and premature aging. These mice demonstrate the fundamental importance of the accumulation of mtDNA alterations in aging. We present here an integrative model where aging is provoked by a single primary event leading to a variety of effects and secondary causes.
Keywords: Mitochondria; Oxidative stress; Mitochondrial respiratory chain; Aging; Life span; DNA damage;

Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase by Pierre Falson; André Goffeau; Marc Boutry; Jean-Michel Jault (133-140).
F1-ATPase, the catalytic sector of Fo-F1 ATPases–ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the α-subunit and Q170Y in the β-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity. The βQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity. In contrast, the αQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking αQ173 to the adjacent βT354, localized at the αDP–βTP interface. The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor α- and β-subunits. Indeed, in the beef heart enzyme, (i) the αDP–βTP interface is stabilized by a vicinal αR171–βD352 salt bridge (ii) βD352 and βT354 belong to a short peptidic stretch close to βY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the βY345–βT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.
Keywords: F1-ATPase; Mitochondria; Negative cooperativity; α-subunit; β-subunit; Schizosaccharomyces pombe; Yeast;

“Wages of Fear”: transient threefold decrease in intracellular ATP level imposes apoptosis by Denis S Izyumov; Armine V Avetisyan; Olga Yu Pletjushkina; Dmitrii V Sakharov; Karel W Wirtz; Boris V Chernyak; Vladimir P Skulachev (141-147).
In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10–35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H2O2-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular “ATP-meter(s)” generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).
Keywords: ATP; Intracellular ATP-meter; Apoptosis; Necrosis; Reactive oxygen species;

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica by Albina Abdrakhmanova; Volker Zickermann; Mihnea Bostina; Michael Radermacher; Hermann Schägger; Stefan Kerscher; Ulrich Brandt (148-156).
Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Accessory subunit; Yarrowia lipolytica; Mass spectrometry;

Activation by retinoids of the uncoupling protein UCP1 by Paula Tomás; Jesús Jiménez-Jiménez; Pilar Zaragoza; Vidyasagar Vuligonda; Roshantha A.S Chandraratna; Eduardo Rial (157-164).
The uncoupling protein from brown adipose tissue (UCP1) is a transporter that catalyzes a regulated discharged of the mitochondrial proton gradient. The proton conductance in UCP1 is inhibited by nucleotides and activated by fatty acids. We have recently shown that all-trans-retinoic acid (ATRA) is a high-affinity activator of UCP1. In the present report, we have set to analyze the structural requirements for the ligands that activate UCP1 and particularly the specificity for different retinoids. For this purpose, we have developed a new protocol to determine the activity of UCP1 in respiring yeast mitochondria that can be adapted for high-throughput screenings. Our results evidence differences between the structural requirements for the activation by fatty acids and retinoids. Thus, although all active retinoids must possess a carboxylate, the introduction of additional polar groups renders them inactive. The linear and rigid structure of these molecules suggests the existence of a long hydrophobic binding pocket. We postulate that the access to the retinoid binding site must occur from the lipid bilayer and this could be at the interface between two transmembrane α-helices.
Keywords: Uncoupling protein; Retinoid; Screening; Respiration; Mitochondrion; Yeast;

The quinone chemistry of bc complexes by Peter R Rich (165-171).
The quinone chemistry that gives rise to the rather unusual strict bifurcation of electron transfer at the Qo site of the cytochrome bc complexes remains controversial. In this article, I review recent ideas and propose a “logic-gated” binding mechanism that combines classical quinone electrochemistry with specific hydrogen bonding requirements and results in a reversible reaction that minimizes unwanted side-reactions that could otherwise undermine the efficiency of the Q-cycle proton/electron coupling mechanism.
Keywords: Cytochrome bc complex; Ubiquinone; Q cycle; Electrochemistry;

Bioenergetics shapes cellular death pathways in Leber's hereditary optic neuropathy: a model of mitochondrial neurodegeneration by Valerio Carelli; Michela Rugolo; Gianluca Sgarbi; Anna Ghelli; Claudia Zanna; Alessandra Baracca; Giorgio Lenaz; Eleonora Napoli; Andrea Martinuzzi; Giancarlo Solaini (172-179).
Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically.In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress.The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.
Keywords: LHON; Mitochondria; Complex I; ATP synthesis; Apoptosis; ROS;