BBA - Bioenergetics (v.1817, #10)
Editorial Board (i).
EBEC 2012: Combining the multiple facets of bioenergetics by Thorsten Friedrich; Oliver Einsle; Peter Gräber (1709-1710).
Rotational catalysis in proton pumping ATPases: From E. coli F-ATPase to mammalian V-ATPase by Masamitsu Futai; Mayumi Nakanishi-Matsui; Haruko Okamoto; Mizuki Sekiya; Robert K. Nakamoto (1711-1721).
We focus on the rotational catalysis of Escherichia coli F-ATPase (ATP synthase, FOF1). Using a probe with low viscous drag, we found stochastic fluctuation of the rotation rates, a flat energy pathway, and contribution of an inhibited state to the overall behavior of the enzyme. Mutational analyses revealed the importance of the interactions among β and γ subunits and the β subunit catalytic domain. We also discuss the V-ATPase, which has different physiological roles from the F-ATPase, but is structurally and mechanistically similar. We review the rotation, diversity of subunits, and the regulatory mechanism of reversible subunit dissociation/assembly of Saccharomyces cerevisiae and mammalian complexes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).►Rotational catalysis of E. coli F-ATPase is discussed. ►We found stochastic rotational behavior including inhibited and active states. ►The flat energy landscape is important in the high efficiency of energy coupling. ►Mutational studies indicated important domain for βγ interaction and catalysis. ►We have discussed our recent studies on V-ATPase.
Keywords: ATP synthase; F-ATPase; V-ATPase; Subunit rotation; Single molecule observation;
Elastic deformations of the rotary double motor of single FoF1-ATP synthases detected in real time by Förster resonance energy transfer by Stefan Ernst; Monika G. Düser; Nawid Zarrabi; Stanley D. Dunn; Michael Börsch (1722-1731).
Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8 nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. FOF1-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary FO motor in the Escherichia coli enzyme is connected to a 3-stepped F1 motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual FOF1-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► FOF1-ATP synthase comprises two rotary motors with different step sizes. ► Coupling of the motors requires reversible elastic deformations. ► Single-molecule FRET is used to measure the elastic angular twist. ► Up to 108° twisting between c- and ε-subunits is found. ► Twisting is suppressed by DCCD during ATP synthesis and ATP hydrolysis.
Keywords: FOF1-ATP synthase; Rotary motor; Elastic deformation; Förster resonance energy transfer; Single-molecule FRET;
Rotary catalysis of the stator ring of F1-ATPase by Ryota Iino; Hiroyuki Noji (1732-1739).
F1-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α3β3 ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F1-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► We observed conformational dynamics of F1-ATPase using high-speed AFM. ► We revealed rotary catalysis of the rotorless α3β3 stator ring of F1-ATPase. ► We proposed new model for the operating principle behind the F1-ATPase.
Keywords: F1-ATPase; Molecular motor; Rotary catalysis; Cooperativity; High-speed atomic force microscopy; Hexameric ATPase;
Effect of mtDNA point mutations on cellular bioenergetics by Joanna Szczepanowska; Dominika Malinska; Mariusz R. Wieckowski; Jerzy Duszynski (1740-1746).
This overview discusses the results of research on the effects of most frequent mtDNA point mutations on cellular bioenergetics. Thirteen proteins coded by mtDNA are crucial for oxidative phosphorylation, 11 of them constitute key components of the respiratory chain complexes I, III and IV and 2 of mitochondrial ATP synthase. Moreover, pathogenic point mutations in mitochondrial tRNAs and rRNAs generate abnormal synthesis of the mtDNA coded proteins. Thus, pathogenic point mutations in mtDNA usually disturb the level of key parameter of the oxidative phosphorylation, i.e. the electric potential on the inner mitochondrial membrane (Δψ), and in a consequence calcium signalling and mitochondrial dynamics in the cell. Mitochondrial generation of reactive oxygen species is also modified in the mutated cells. The results obtained with cultured cells and describing biochemical consequences of mtDNA point mutations are full of contradictions. Still they help elucidate the biochemical basis of pathologies and provide a valuable tool for finding remedies in the future. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Keywords: mtDNA point mutation; Mitochondrial disease; Mitochondrial membrane potential; Mitochondrial calcium; ROS; Mitochondrial dynamics;
Complex effects of 17β-estradiol on mitochondrial function by Anika Thiede; Frank Norbert Gellerich; Peter Schönfeld; Detlef Siemen (1747-1753).
Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17β-estradiol (βE) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca2 +-induced permeability transition, as well, though only at higher concentrations (≥ 30 μM). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that βE diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that βE affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Keywords: Mitochondria; 17 β-estradiol; Permeability transition pore; Mitochondrial BK-channel; Mitochondrial respiration;
New perspectives on assembling c-type cytochromes, particularly from sulphate reducing bacteria and mitochondria by Stuart J. Ferguson (1754-1758).
Some recent new developments emerging from studies of the Systems I and III for c-type cytochrome biogenesis are discussed, particularly in regard to developments in studying System I in sulphate reducing bacteria. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► The Ccm system (System I) is a multicomponent system for attaching heme to the thiol groups of two cysteines on apoproteins. ► A novel cross linked by heme product between a component of the Ccm system (E) and an apocytochrome is discussed. ► Progress in understanding how System III recognises its target apocytochrome is summarised. ► A highly conserved phenylalanine is crucial for interaction between System III and its target. ► Sulphate reducing bacteria contain a variant of System I that poses many issues.
Keywords: Cytochrome c assembly; Heme synthesis via siroheme;
A role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial uncoupling protein 1 (UCP1) by Kieran J. Clarke; Alison E. Adams; Lars H. Manzke; Terry W. Pearson; Christoph H. Borchers; Richard K. Porter (1759-1767).
In this study we show that mitochondrial uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and thymus mitochondria can be ubiquitinylated and degraded by the cytosolic proteasome. Using a ubiquitin conjugating system, we show that UCP1 can be ubiquitinylated in vitro. We demonstrate that UCP1 is ubiquitinylated in vivo using isolated mitochondria from brown adipose tissue, thymus and whole brown adipocytes. Using an in vitro ubiquitin conjugating–proteasome degradation system, we show that the cytosolic proteasome can degrade UCP1 at a rate commensurate with the half-life of UCP1 (i.e. 30–72 h in brown adipocytes and ~ 3 h, in thymocytes). In addition, we demonstrate that the cytoplasmic proteasome is required for UCP1 degradation from mitochondria that the process is inhibited by the proteasome inhibitor MG132 and that dissipation of the mitochondrial membrane potential inhibits degradation of UCP1. There also appears to be a greater amount of ubiquitinylated UCP1 associated with BAT mitochondria from cold-acclimated animals. We have also identified (using immunoprecipitation coupled with mass spectrometry) ubiquitinylated proteins with molecular masses greater than 32 kDa, as being UCP1. We conclude that there is a role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial UCP1. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► UCP1 is ubiquitinylated in mitochondria from brown adipose tissue and thymus. ► Ubiquitinylated UCP1 is degraded by the cytosolic proteasome. ► The half-life of UCP1 in thymocytes is approximately 3 h. ► Cold-acclimation increases the amount of UCP1 that is ubiquitinylated in BAT.
Keywords: Mitochondria; Ubiquitinylation; Proteosome; UCP1; Thymus; Brown adipose tissue;
Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation by M. Mar González-Barroso; Andrea Anedda; Eunate Gallardo-Vara; Mariano Redondo-Horcajo; Leonor Rodríguez-Sánchez; Eduardo Rial (1768-1775).
While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis. While AMP/ATP ratios were unaffected by metformin, phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase augmented. The inhibition of respiration provoked a rapid and sustained increase in superoxide levels, despite the increase in UCP2 and superoxide dismutase activity. The inhibition of respiration was rapidly reversed by fatty acids and thus respiration was lower in treated cells in the presence of pyruvate and glucose while rates were identical to control cells when palmitate was the substrate. We conclude that metformin reversibly inhibits mitochondrial respiration, it rapidly activates AMPK without altering the energy charge, and it inhibits fatty acid synthesis. Mitochondrial β-oxidation is facilitated by reversing the inhibition of complex I and, presumably, by releasing the inhibition of carnitine palmitoyltransferase. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Metformin reversibly inhibits mitochondrial respiration in 3T3-L1 adipocytes. ► Inhibition of respiration leads to the rapid activation of AMPK. ► AMPK activation occurs without changes in adenine nucleotide levels. ► Fatty acids revert the inhibition of complex I to facilitate their oxidation.
Keywords: Metformin; Diabetes; Adipocyte; Fatty acid oxidation; Mitochondria; AMPK;
Tracing the tail of ubiquinone in mitochondrial complex I by Heike Angerer; Hamid R. Nasiri; Vanessa Niedergesäß; Stefan Kerscher; Harald Schwalbe; Ulrich Brandt (1776-1784).
Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron–sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30 Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).Display Omitted► Ubiquinone interactions of complex I were analyzed by site-directed mutagenesis. ► Five ubiquinone derivatives with different tails were used for functional analysis. ► The isoprenoid tail structure is critical for access to the ubiquinone active site. ► A methionine rich domain acts as access gate for the hydrophobic ubiquinone tail.
Keywords: Respiratory chain; Mitochondria; Complex I; Ubiquinone; Rotenone; Yarrowia lipolytica;
The coupling mechanism of respiratory complex I — A structural and evolutionary perspective by Rouslan G. Efremov; Leonid A. Sazanov (1785-1795).
Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).Display Omitted► Based on structure and conservation, the coupling site of complex I is localized. ► Coupling mechanism of complex I-related enzymes is conserved. ► A model of coupling mechanism, common for complex I and hydrogenases, is suggested.
Keywords: NADH:ubiquinone oxidoreductase; Hydrogenase; Modular evolution; Coupling mechanism; Redox reaction; Proton translocation;
Mitochondrial and ion channel gene alterations in autism by Moyra Smith; Pamela L. Flodman; John J. Gargus; Mariella T. Simon; Kimberley Verrell; Richard Haas; Gail E. Reiner; Robert Naviaux; Katherine Osann; M. Anne Spence; Douglas C. Wallace (1796-1802).
To evaluate the potential importance in autistic subjects of copy number variants (CNVs) that alter genes of relevance to bioenergetics, ionic metabolism, and synaptic function, we conducted a detailed microarray analysis of 69 autism probands and 35 parents, compared to 89 CEU HapMap controls. This revealed that the frequency CNVs of ≥ 100 kb and CNVs of ≥ 10 Kb were markedly increased in probands over parents and in probands and parents over controls. Evaluation of CNVs ≥ 1 Mb by chromosomal FISH confirmed the molecular identity of a subset of the CNVs, some of which were associated with chromosomal rearrangements. In a number of the cases, CNVs were found to alter the copy number of genes that are important in mitochondrial oxidative phosphorylation (OXPHOS), ion and especially calcium transport, and synaptic structure. Hence, autism might result from alterations in multiple bioenergetic and metabolic genes required for mental function. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► CNVs are significantly increased in autism. ► Some autism patients have systemic OXPHOS defects. ► CNVs often encompass mitochondrial, ion channel, and synaptic genes.
Keywords: Autism; Mitochondria; OXPHOS; Copy number variants (CNVs); Calcium channel; Synapses;
EPR detection of two protein-associated ubiquinone components (SQNf and SQNs) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I by Tomoko Ohnishi; S. Tsuyoshi Ohnishi; Kyoko Shinzawa-Itoh; Shinya Yoshikawa; Ralph T. Weber (1803-1809).
The success of Sazanov's group in determining the X-ray structure of the whole bacterial complex I is a great contribution to the progress of complex I research. In this mini-review of 35 years' history of my laboratory and collaborators, we characterized the function of protein-associated semiquinone molecules in the proton-pumping mechanism in complex I (NADH-quinone oxidoreductase). We have constructed most of the frame work of our hypothesis, utilizing EPR techniques before the X-ray structures of complex I were reported by Sazanov's and Brandt's groups. One of the semiquinones (SQNf) is extremely sensitive to a proton motive force imposed on the energy-transducing membrane, while the other (SQNs) is insensitive. Their sensitivity to rotenone inhibition also differs. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (> 8). We determined the distance between SQNf and iron–sulfur cluster N2 on the basis of their direct spin–spin interaction. We are extending this line of work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio > 5. Two frontier research groups support our view point based on their mutagenesis studies. High frequency (33.9 GHz; Q-band) EPR experiments appear to favor our two-semiquinone model. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► In many cases, two semiquinones are required to couple electron and proton transfer. ► In complex I, the fast relaxing semiquinone is sensitive to proton motive force. ► The semiquinone is spin-coupled with N2, and may function in “direct” proton pumping. ► In complex I, the slow relaxing semiquinone is insensitive to proton motive force. ► This semiquinone may transport scalar proton and trigger “indirect” proton transfer.
Keywords: EPR; SQ-Nf and SQ-Ns; Membrane in situ; Proteoliposome; Bovine heart complex I; Proton-pumping mechanism;
Study of ion translocation by respiratory complex I. A new insight using 23Na NMR spectroscopy by Ana P. Batista; Bruno C. Marreiros; Ricardo O. Louro; Manuela M. Pereira (1810-1816).
The research on complex I has gained recently a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism(s). The proton has been identified as the coupling ion, although in the case of some bacterial complexes I Na+ has been proposed to have that role. We have addressed the relation of some complexes I with Na+ and developed an innovative methodology using 23Na NMR spectroscopy. This allowed the investigation of Na+ transport taking the advantage of directly monitoring changes in Na+ concentration. Methodological aspects concerning the use of 23Na NMR spectroscopy to measure accurately sodium transport in bacterial membrane vesicles are discussed here. External-vesicle Na+ concentrations were determined by two different methods: 1) by integration of the resonance frequency peak and 2) using calibration curves of resonance frequency shift dependence on Na+ concentration. Although the calibration curves are a suitable way to determine Na+ concentration changes under conditions of fast exchange, it was shown not to be applicable to the bacterial membrane vesicle systems. In this case, the integration of the resonance frequency peak is the most appropriate analysis for the quantification of external-vesicle Na+ concentration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).►The relation of complex I and Na+ was previously studied by an original approach. ►We developed an innovative methodology using 23Na NMR spectroscopy. ►The method had the advantage of directly monitoring changes in Na+ concentration. ►Integration of the resonance frequency peak is most accurate for Na+ quantification.
Keywords: Complex I; NADH:quinone oxidoreductase; 23Na NMR spectroscopy; Resonance frequency shift; Shift reagent,Tm(DOTP)5 −;
The single NqrB and NqrC subunits in the Na+-translocating NADH: Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae each carry one covalently attached FMN by Marco S. Casutt; Andreas Schlosser; Wolfgang Buckel; Julia Steuber (1817-1822).
The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe–2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na+-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na+-NQR contains approximately 1.7 mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na+-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na+-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).Display Omitted► The Na+ -translocating NADH:quinone oxidoreductase is composed of six subunits. ► Each NqrB and NqrC subunit carries one riboflavin phosphate bound to threonine. ► Phosphodiester-linked flavins are released by β-elimination with LiOH. ► The threonine residues in the flavin binding sites are identified by mass spectroscopy.
Keywords: Covalent flavin cofactor; Protein modification; Respiratory sodium pump; NADH dehydrogenase; Na+ transport;
Insights into the mechanism of electron transfer and sodium translocation of the Na+-pumping NADH:quinone oxidoreductase by Oscar Juárez; Blanca Barquera (1823-1832).
Na+-NQR is a unique energy-transducing complex, widely distributed among marine and pathogenic bacteria. It converts the energy from the oxidation of NADH and the reduction of quinone into an electrochemical Na+-gradient that can provide energy for the cell. Na+-NQR is not homologous to any other respiratory protein but is closely related to the RNF complex. In this review we propose that sodium pumping in Na+-NQR is coupled to the redox reactions by a novel mechanism, which operates at multiple sites, is indirect and mediated by conformational changes of the protein. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Na+-NQR is a respiratory enzyme essential in the metabolism of many marine and pathogenic bacteria. ► Na+-NQR selectively translocates only Na+ across the cell membrane. ► Na+-NQR has five redox cofactors: FAD, 2Fe-2S center, two covalently bound FMN's and riboflavin. ► Coupling of Na+ transport to redox reactions is indirect. ► Coupling is likely mediated by conformational changes.
Keywords: Na+-NQR; Primary sodium pump; NADH:quinone oxidoreductase; Na+ translocation; Bacterial energy transduction;
Bioenergetic role of mitochondrial fusion and fission by Benedikt Westermann (1833-1838).
Mitochondria are highly dynamic organelles. Frequent cycles of fusion and fission adapt the morphology of the mitochondrial compartment to the metabolic needs of the cell. Mitochondrial fusion is particularly important in respiratory active cells. It allows the spreading of metabolites, enzymes, and mitochondrial gene products throughout the entire mitochondrial compartment. This serves to optimize mitochondrial function and counteracts the accumulation of mitochondrial mutations during aging. Fragmented mitochondria are frequently found in resting cells, and mitochondrial fission plays an important role in the removal of damaged organelles by autophagy. Thus, mitochondrial fusion and fission both contribute to maintenance of mitochondrial function and optimize bioenergetic capacity. Multiple signalling pathways regulate the machinery of mitochondrial dynamics to adapt the shape of the mitochondrial compartment to the metabolic conditions of the cell. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Mitochondrial dynamics and morphology reflect respiratory activity. ► Mitochondrial fusion is required for maximum respiratory capacity. ► Mitochondrial fission plays a role in degradation of dysfunctional organelles.
Keywords: Aging; Mitochondrial dynamics; Mitochondrial morphology; Respiratory capacity;
A review of the binding-change mechanism for proton-translocating transhydrogenase by J. Baz Jackson (1839-1846).
Proton-translocating transhydrogenase is found in the inner membranes of animal mitochondria, and in the cytoplasmic membranes of many bacteria. It catalyses hydride transfer from NADH to NADP+ coupled to inward proton translocation. Evidence is reviewed suggesting the enzyme operates by a “binding-change” mechanism. Experiments with Escherichia coli transhydrogenase indicate the enzyme is driven between “open” and “occluded” states by protonation and deprotonation reactions associated with proton translocation. In the open states NADP+/NADPH can rapidly associate with, or dissociate from, the enzyme, and hydride transfer is prevented. In the occluded states bound NADP+/NADPH cannot dissociate, and hydride transfer is allowed. Crystal structures of a complex of the nucleotide-binding components of Rhodospirillum rubrum transhydrogenase show how hydride transfer is enabled and disabled at appropriate steps in catalysis, and how release of NADP+/NADPH is restricted in the occluded state. Thermodynamic and kinetic studies indicate that the equilibrium constant for hydride transfer on the enzyme is elevated as a consequence of the tight binding of NADPH relative to NADP+. The protonation site in the translocation pathway must face the outside if NADP+ is bound, the inside if NADPH is bound. Chemical shift changes detected by NMR may show where alterations in protein conformation resulting from NADP+ reduction are initiated. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Transhydrogenase couples the reduction of NADP+ by NADH to proton translocation. ► Coupling is probably achieved by a binding-change mechanism. ► Protonation/deprotonation interconverts the enzyme between open and occluded states. ► Nucleotide binding, proton access and hydride transfer are switched during catalysis. ► Experimental evidence for the binding-change mechanism is reviewed.
Keywords: Transhydrogenase; Proton translocation; Membrane protein; Nucleotide binding; X-ray crystallography; NMR;
Fusing proteins as an approach to study bioenergetic enzymes and processes by Monika Czapla; Marcin Sarewicz; Artur Osyczka (1847-1851).
Fusing proteins is an attractive genetic tool used in several biochemical and biophysical investigations. Within a group of redox proteins, certain fusion constructs appear to provide valuable templates for spectroscopy with which specific bioenergetic questions can be addressed. Here we briefly summarize three different cases of fusions reported for bacterial cytochrome bc 1 (prokaryotic equivalent of mitochondrial respiratory complex III), a common component of electron transport chains. These fusions were used to study supramolecular organization of enzymatic complexes in bioenergetic membrane, influence of the accessory subunits on the activity and stability of the complex, and molecular mechanism of operation of the enzyme in the context of its dimeric structure. Besides direct connotation to molecular bioenergetics, these fusions also appeared interesting from the protein design, biogenesis, and assembly points of view. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Cytochrome bc 1 is capable of accommodating various types of fusions. ► Fused forms of cytochrome bc 1 are used to address specific bioenergetic questions. ► Cases of fusion reflect structural plasticity of cytochrome bc 1.
Keywords: Fusion protein; Protein design; Electron transfer; Cytochrome; Membrane protein;
The Alternative complex III: Properties and possible mechanisms for electron transfer and energy conservation by Patrícia N. Refojo; Miguel Teixeira; Manuela M. Pereira (1852-1859).
Alternative complexes III (ACIII) are recently identified membrane-bound enzymes that replace functionally the cytochrome bc 1/ b 6 f complexes. In general, ACIII are composed of four transmembrane proteins and three peripheral subunits that contain iron–sulfur centers and C-type hemes. ACIII are built by a combination of modules present in different enzyme families, namely the complex iron–sulfur molybdenum containing enzymes. In this article a historical perspective on the investigation of ACIII is presented, followed by an overview of the present knowledge on these enzymes. Electron transfer pathways within the protein are discussed taking into account possible different locations (cytoplasmatic or periplasmatic) of the iron–sulfur containing protein and their contribution to energy conservation. In this way several hypotheses for energy conservation modes are raised including linear and bifurcating electron transfer pathways. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► ACIII are quinol: electron acceptors oxidoreductases. ► The orientation of the peripheral subunit ActB is discussed. ► Electron transfer in ACIII may be linear or bifurcated. ► ACIII may contribute to energy conservation.
Keywords: Alternative Complex III; Electron bifurcation; Energy conservation; Quinol/quinone; bc1/b6f complex;
Induction of the permeability transition pore in cells depleted of mitochondrial DNA by Ionica Masgras; Andrea Rasola; Paolo Bernardi (1860-1866).
Respiratory complexes are believed to play a role in the function of the mitochondrial permeability transition pore (PTP), whose dysregulation affects the process of cell death and is involved in a variety of diseases, including cancer and degenerative disorders. We investigated here the PTP in cells devoid of mitochondrial DNA (ρ0 cells), which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions. We observed that mitochondria of ρ0 cells maintain a membrane potential and that this is readily dissipated after displacement of hexokinase (HK) II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by placing ρ0 cells in a medium without serum and glucose. The PTP inhibitor cyclosporin A (CsA) could decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. We also found that a fraction of the kinases ERK1/2 and GSK3α/β is located in the mitochondrial matrix of ρ0 cells, and that glucose and serum deprivation caused concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of cyclophilin D, the mitochondrial target of CsA. GSK3α/β inhibition with indirubin-3′-oxime decreased PTP-induced cell death in ρ0 cells following nutrient ablation. These findings indicate that ρ0 cells are equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells, and suggest that escape from PTP opening is a survival factor in this model of mitochondrial diseases. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► A mitochondrial (mt) permeability transition (PT) occurs in human 206 ρ0 cells. ► Detachment of mt hexokinase II is an effective PT trigger. ► A fraction of ERK1/2 and GSK3α/β is in the mt matrix of ρ0 cells. ► Glucose/serum deprivation (GSD) inhibits mt ERK and activates mt GSK3α/β. ► PT plays a role in the death of 206 ρ0 cells by GSD.
Keywords: Mitochondrion; ρ0 cell; Permeability transition; Cyclophilin D; Hexokinase; GSK3β;
A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus by Anna Kajma; Adam Szewczyk (1867-1878).
Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288 ± 4 pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150 mM KCl). At positive voltages, this channel displayed a conductance of 67.84 pS and a strong voltage dependence at holding potentials from − 80 mV to + 80 mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Two potassium channels were identify in embryonic rat hippocampus mitochondria. ► Low conductance potassium channel was regulated by pH. ► This channel does not correspond to any previously described potassium channels in mitochondria.
Keywords: Mitochondria; Potassium channels; Brain ischemia;
Mitochondrial hydrogen peroxide production as determined by the pyridine nucleotide pool and its redox state by Alexandra V. Kareyeva; Vera G. Grivennikova; Andrei D. Vinogradov (1879-1885).
The rates of NADH-supported superoxide/hydrogen peroxide production by membrane-bound bovine heart respiratory complex I, soluble pig heart dihydrolipoamide dehydrogenase (DLDH), and by accompanying operation of these enzymes in rat heart mitochondrial matrix were measured as a function of the pool of pyridine nucleotides and its redox state. Each of the activities showed nontrivial dependence on nucleotide pool concentration. The NAD+/NADH ratios required for their half maximal capacities were determined. About half of the total NADH-supported H2O2 production by permeabilized mitochondria in the absence of stimulating ammonium could be accounted for by DLDH activity. The significance of the mitochondrial NADH-dependent hydrogen peroxide production under physiologically relevant conditions is discussed. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Mitochondrial ROS production is a function of NADH/NAD+ pool and its redox state. ► Complex I and dihydrolipoamide dehydrogenase (DLDH) generate similar amounts of ROS. ► Up to 90% of ammonium activated ROS production is due to DLDH activity. ► At physiological conditions either enzyme operates at 20% of its potential capacity.
Keywords: Respiratory complex I; Dihydrolipoamide dehydrogenase; Hydrogen peroxide; Pyridine nucleotides; Mitochondria;
The antiapoptotic OPA1/Parl couple participates in mitochondrial adaptation to heat shock by Luiza K. Sanjuán Szklarz; Luca Scorrano (1886-1893).
The mitochondria-shaping protein optic atrophy 1 (OPA1) has genetically distinguishable roles in mitochondrial morphology and apoptosis. The latter depends on the presenilin associated rhomboid like (PARL) protease, essential for the accumulation of a soluble intermembrane space form of OPA1 (IMS-OPA1). Here we show that OPA1 and PARL participate in the heat shock response, a stereotypical cellular process of adaptation to thermal stress. Upon heat shock, long forms of OPA1 are lost and mitochondria fragment. However, mitochondrial fusion is dispensable to maintain viability, whereas IMS-OPA1 is required. Upon conditioning—a process of mild heat shock and recovery—IMS-OPA1 accumulates, OPA1 oligomers increase and mitochondria release less cytochrome c, ultimately resulting in cellular resistance to subsequent apoptotic inducers. In Parl −/− cells accumulation of IMS-OPA1 is blunted and conditioning fails to protect from cytochrome c release and apoptosis. Thus, the OPA1/PARL dependent pathway of cristae remodeling is implicated in heat shock. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Heat shock causes cleavage of the cristae regulator OPA1. ► Heat shock causes PARL-dependent accumulation of soluble antiapoptotic OPA1. ► Soluble OPA1 confers secondary resistance to apoptosis to heat shocked cells. ► OPA1 is essential also for heat shock conditioning.
Keywords: Mitochondrion; OPA1; PARL; Heat shock response; Cytochrome c release; Apoptosis;
Product-controlled steady-state kinetics between cytochrome aa 3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach by Myat T. Lin; Robert B. Gennis (1894-1900).
Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c 2 +) as the electron donor. In this study, the oxidation of horse cyt c 2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c 2 + and cyt c 3 +. This allowed an analysis of the effects of cyt c 3 + on the steady-state kinetics between CcO and cyt c 2 +. The results show that cyt c 3 + exhibits product inhibition by two mechanisms: competition with cyt c 2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c 2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).Display Omitted► Steady state kinetics of R. sphaeroides cyt c oxidase and horse cyt c is monophasic. ► Full spectrum deconvolution used to obtain [cyt c 2+] and [cyt c 3+] at each point. ► Analysis shows product inhibition by cyt c 3+ due to interaction at two sites. ► cyt c 3+ is a competitive inhibitor at the catalytic site. ► cyt c 3+ also binds at a second, allosteric site and modulates both KM and Vmax.
Keywords: Cytochrome c oxidase; Cytochrome c; Kinetics;
The contribution of thioredoxin-2 reductase and glutathione peroxidase to H2O2 detoxification of rat brain mitochondria by Alexei P. Kudin; Bartłomiej Augustynek; Anja Kerstin Lehmann; Richard Kovács; Wolfram S. Kunz (1901-1906).
Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H2O2) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60 ± 20% and 20 ± 15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► We determined the role of Trx2-, GSH-systems and catalase for H2O2 detoxification. ► The contribution of the Trx2- and GSH-systems was in rat brain 60 ± 20% and 20 ± 15%. ► The Trx2- and GSH-systems predominantly detoxify mitochondrially produced H2O2. ► Interneurons and microglia showed strong response to inhibition of Trx2-, GSH-systems. ► Pyramidal cells and astrocytes showed weak response to inhibition of Trx2-, GSH-systems.
Keywords: Rat brain mitochondria; Hydrogen peroxide metabolism; Thioredoxin-2 reductase; Glutathione peroxidase;
Structure and function of bacterial nitric oxide reductases by Yoshitsugu Shiro (1907-1913).
The crystal structures of bacterial nitric oxide reductases (NOR) from Pseudomonas aeruginosa and Geobacillus stearothermophilus were reported. The structural characteristics of these enzymes, especially at the catalytic site and on the pathway that catalytic protons are delivered, are compared, and the corresponding regions of aerobic and micro-aerobic cytochrome oxidases, O2 reducing enzymes, which are evolutionarily related to NOR are discussed. On the basis of these structural comparisons, a mechanism for the reduction of NO to produce N2O by NOR, and the possible molecular evolution of the proton pumping ability of the respiratory enzymes is discussed. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Overall structures are basically similar between two NORs, cNOR and qNOR. ► Direction for the catalytic proton transfer is entirely different. ► Based on structural comparison, a possible NO reduction mechanism can be proposed.
Keywords: NO reductase; Cytochrome oxidase; Respiratory enzyme; Denitrification; Greenhouse gas; Ozone depletion;
Proton transfer in the quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus during reduction of oxygen by Lina Salomonsson; Joachim Reimann; Takehiko Tosha; Nils Krause; Nathalie Gonska; Yoshitsugu Shiro; Pia Ädelroth (1914-1920).
Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246]. In this study, we have investigated the reaction between this qNOR and oxygen. Our results show that, like some cNORs, the G. stearothermophilus qNOR is capable of O2 reduction with a turnover of ~ 3 electrons s− 1 at 40 °C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) qNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between qNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in qNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Quinol-dependent nitric oxide reductase from G. stearothermophilus can reduce O2. ► The fully reduced qNOR is oxidised by O2 in a flow-flash experiment. ► A proton-coupled electron transfer with time constant = 1.8 ms is observed. ► The pH dependence of this phase is different from cNOR. ► Possible proton transfer pathways in qNOR are discussed.
Keywords: Heme-copper oxidase; Proton transfer pathway; Non-heme iron; Flow-flash; Carbon monoxide;
Assignment of the CO-sensitive carboxyl group in mitochondrial forms of cytochrome c oxidase using yeast mutants by Amandine Maréchal; Brigitte Meunier; Peter R. Rich (1921-1924).
Point mutations of E243D and I67N were introduced into subunit I of a 6histidine-tagged (6H-WT) form of yeast Saccharomyces cerevisiae mitochondrial cytochrome c oxidase. The two mutants (6H-E243DI and 6H-I67NI) were purified and showed ≈ 50 and 10% of the 6H-WT turnover number. Light-induced CO photolysis FTIR difference spectra of the 6H-WT showed a peak/trough at 1749/1740 cm− 1, as seen in bovine CcO, which downshifted by 7 cm− 1 in D2O. The bands shifted to 1736/1762 cm− 1 in 6H-E243DI, establishing that the carboxyl group affected by CO binding in mitochondrial CcOs is E243. In 6H-I67NI, the trough at 1740 cm− 1 was shifted to 1743 cm− 1 and its accompanying peak intensity was greatly reduced. This confirms that the I67N mutation interferes with conformational alterations around E243. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► CO photolysis from yeast CcO induces the same carboxyl change seen in bovine CcO. ► E243D and I67N mutants were analysed to identify the responsible amino acid. ► Changes in the IR spectra definitively identify E243 as the CO-sensitive amino acid. ► The I67N mutation inhibits the enzyme by interfering with E243 function.
Keywords: Mitochondria; Cytochrome c oxidase; FTIR; Site-directed mutagenesis; Yeast;
Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts by Federica Valsecchi; Claire Monge; Marleen Forkink; Ad J.C. de Groof; Giovanni Benard; Rodrigue Rossignol; Herman G. Swarts; Sjenet E. van Emst-de Vries; Richard J. Rodenburg; Maria A. Calvaruso; Leo G.J. Nijtmans; Bavo Heeman; Peggy Roestenberg; Be Wieringa; Jan A.M. Smeitink; Werner J.H. Koopman; Peter H.G.M. Willems (1925-1936).
Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (FoF1-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~ 830 kDa (enzymatically inactive) and ~ 200 kDa (active). The level of fully-assembled CII–CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O2 consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O2 consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD+]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP+]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► MEFs from NDUFS4 knockout animals contain an active but destabilized complex I. ► NDUFS4 gene deletion triggers a slightly more glycolytic cellular phenotype. ► NDUFS4 gene deletion does not increase ROS levels. ► NDUFS4 gene deletion does not detectably affect total and free cytosolic ATP levels.
Keywords: Metabolic disorder; Oxidative phosphorylation; Respirometry; Live-cell microscopy; Glycolysis;
Cardiolipin binding in bacterial respiratory complexes: Structural and functional implications by Rodrigo Arias-Cartin; Stéphane Grimaldi; Pascal Arnoux; Bruno Guigliarelli; Axel Magalon (1937-1949).
The structural and functional integrity of biological membranes is vital to life. The interplay of lipids and membrane proteins is crucial for numerous fundamental processes ranging from respiration, photosynthesis, signal transduction, solute transport to motility. Evidence is accumulating that specific lipids play important roles in membrane proteins, but how specific lipids interact with and enable membrane proteins to achieve their full functionality remains unclear. X-ray structures of membrane proteins have revealed tight and specific binding of lipids. For instance, cardiolipin, an anionic phospholipid, has been found to be associated to a number of eukaryotic and prokaryotic respiratory complexes. Moreover, polar and septal accumulation of cardiolipin in a number of prokaryotes may ensure proper spatial segregation and/or activity of proteins. In this review, we describe current knowledge of the functions associated with cardiolipin binding to respiratory complexes in prokaryotes as a frame to discuss how specific lipid binding may tune their reactivity towards quinone and participate to supercomplex formation of both aerobic and anaerobic respiratory chains. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Cardiolipin is a key component of energy-transducing membranes. ► Cardiolipin tunes the reactivity of the nitrate reductase complex towards quinone. ► Cardiolipin may influence quinone reactivity of the formate dehydrogenase complex. ► Polar and septal accumulation of cardiolipin is observed in bacteria. ► Cardiolipin binding may be at the origin of supercomplex formation in bacteria.
Keywords: Cardiolipin; Bacteria; Respiratory complex; Nitrate reductase; Formate dehydrogenase; Aerobic and anaerobic respiration;
Electrochemical and infrared spectroscopic analysis of the interaction of the CuA domain and cytochrome c 552 from Thermus thermophilus by Yashvin Neehaul; Ying Chen; Carolin Werner; James A. Fee; Bernd Ludwig; Petra Hellwig (1950-1954).
The hydrophobically guided complex formation between the CuA fragment from Thermus thermophilus ba 3 terminal oxidase and its electron transfer substrate, cytochrome c 552, was investigated electrochemically. In the presence of the purified CuA fragment, a clear downshift of the c 552 redox potential from 171 to 111 mV ± 10 mV vs SHE′ was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Protein–protein interactions may shift redox potentials and thus rule electron transfer. ► The CuA-C552 interaction induced a 90 mV downshift. ► Acidic residues participate to the regulation of the redox potentials.
Keywords: Protein–protein interaction; Electron transfer; Cytochrome c 552; CuA fragment; Electrochemistry; FTIR spectroscopy;
Recent progress in biological charge transfer: Theory and simulation by Thorsten Koslowski; Fabian Burggraf; Sebastian Krapf; Thomas Steinbrecher; Christian Wittekindt (1955-1957).
In this contribution, we discuss three recent developments in atomistic biological charge transfer theory. First, in the context of Marcus' classical theory of charge transfer, key quantities of the theory such as driving forces and reorganization enthalpies are now accessible by thermodynamic integration schemes within standard molecular dynamics simulations at high accuracy. Second, direct simulations of charge transfer enable the computation of fast charge transfer reaction rates without having to resort to Marcus' theory. Finally, exploring the electronic structure beyond that of hitherto presumed centers of localization helps to identify new stepping stones of charge transfer reactions. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).► Recent developments extend the range of charge transfer theory. ► Key energies can be obtained from thermodynamic integration schemes. ► Direct simulations provide a check on Marcus' theory. ► New stepping stones in charge transfer are identified.
Keywords: Charge transfer; Electron transfer; Theory; Simulation; Molecular dynamics;
From in silico to in spectro kinetics of respiratory complex I by Stéphane Ransac; Margit Heiske; Jean-Pierre Mazat (1958-1969).
An enzyme's activity is the consequence of its structure. The stochastic approach we developed to study the functioning of the respiratory complexes is based upon their 3D structure and their physical and chemical properties. Consequently it should predict their kinetic properties. In this paper we compare the predictions of our stochastic model derived for the complex I with a number of experiments performed with a large range of complex I substrates and products. A good fit was found between the experiments and the prediction of our stochastic approach. We show that, due to the spatial separation of the two half redox reactions (NADH/NAD and Q/QH2), the kinetics cannot necessarily obey a simple mechanism (ordered or ping-pong for instance). A plateau in the kinetics is observed at high substrates concentrations, well evidenced in the double reciprocal plots, which is explained by the limiting rate of quinone reduction as compared with the oxidation of NADH at the other end of complex I. Moreover, we show that the set of the seven redox reactions in between the two half redox reactions (NADH/NAD and Q/QH2) acts as an electron buffer. An inhibition of complex I activity by quinone is observed at high concentration of this molecule, which cannot be explained by a simple stochastic model based on the known structure. We hypothesize that the distance between the catalytic site close to N2 (iron/sulfur redox center that transfers electrons to quinone) and the membrane forces the quinone/quinol to take several positions in between these sites. We represent these possible positions by an extra site necessarily occupied by the quinone/quinol molecules on their way to the redox site. With this hypothesis, we are able to fit the kinetic experiments over a large range of substrates and products concentrations. The slow rate constants derived for the transition between the two sites could be an indication of a conformational change of the enzyme during the quinone/quinol movement. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).Display Omitted► Stochastic simulation (Gillespie) of complex I kinetics. ► Experimental kinetics over a large concentration range of substrates and products. ► Good fit between model and experiments. ► Determination of substrates and products binding and release rate constants. ► Quinone inhibition is accounted for by the hypothesis of a second quinone site.
Keywords: Complex I; NADH–ubiquinone oxidoreductase; Stochastic modeling; Inhibition;