BBA - Bioenergetics (v.1555, #1-3)
Editorial Board (ii).
Multidrug transporters and antibiotic resistance in Lactococcus lactis by Gerrit J Poelarends; Piotr Mazurkiewicz; Wil N Konings (1-7).
The Gram-positive bacterium Lactococcus lactis produces two distinct multidrug transporters, designated LmrA and LmrP, that both confer resistance to a wide variety of cationic lipophilic cytotoxic compounds as well as to many clinically relevant antibiotics. While LmrP is a proton/drug antiporter that belongs to the major facilitator superfamily of secondary transporters, LmrA is an ATP-dependent primary transporter that belongs to the ATP-binding cassette superfamily of transport proteins. Both LmrA and LmrP function as “hydrophobic vacuum cleaners” by excreting lipophilic cationic compounds from the inner leaflet of the membrane directly into the external water phase. LmrA is both functionally and structurally homologous to the human multidrug transporter P-glycoprotein. LmrA is a half ABC transporter that is functional as a homodimer, consistent with the general four-domain organization of ABC transporters, and is proposed to mediate drug transport by an alternating two-site transport mechanism.
Keywords: Multidrug resistance; Lactococcus lactis; Antibiotic resistance; Excretion; ABC transporter;
A conformational change in the isolated NADP(H)-binding component (dIII) of transhydrogenase induced by low pH: a reflection of events during proton translocation by the complete enzyme? by Daniel J Rodrigues; J.Baz Jackson (8-13).
Transhydrogenase couples the reduction of NADP+ by NADH to inward proton translocation across the bacterial (or mitochondrial) membrane. Conformational changes in the NADP(H)-binding component of the enzyme (dIII) are central to the coupling mechanism. In the “open” state, NADP(H) bound to dIII can readily exchange with nucleotides in the solvent but hydride transfer [to/from NAD(H) bound to dI] is prevented. In the “occluded” state, bound NADP(H) cannot exchange with solvent nucleotides but the hydride transfer reaction is permitted. It was previously found that the conformational state of isolated, recombinant dIII is pH dependent. At neutral pH, the protein adopts a conformation resembling the occluded state, and at low pH, it adopts a conformation resembling the open state. The crystal structure of dIII indicates that the loop E “lid” might be largely responsible for the very high affinity of the protein for NADP(H). In this paper we show, using fluorescence resonance energy transfer, that the distance between the apex of loop E of isolated dIII, and the core of the protein, increases when the solution pH is lowered. This is consistent with the view that the lid is retracted to permit NADPH release during turnover of the complete enzyme.
Keywords: Transhydrogenase; Proton translocation; Fluorescence resonance energy transfer; Redox protein;
Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention by Peter L Pedersen; Saroj Mathupala; Annette Rempel; J.F Geschwind; Young Hee Ko (14-20).
Despite more than 75 years of research by some of the greatest scientists in the world to conquer cancer, the clear winner is still cancer. This is reflected particularly by liver cancer that worldwide ranks fourth in terms of mortality with survival rates of no more than 3–5%. Significantly, one of the earliest discovered hallmarks of cancer had its roots in Bioenergetics as many tumors were found in the 1920s to exhibit a high glycolytic phenotype. Although research directed at unraveling the underlying basis and significance of this phenotype comprised the focus of cancer research for almost 50 years, these efforts declined greatly from 1970 to 1990 as research into the molecular and cell biology of this disease gained center stage. Certainly, this change was necessary as the new knowledge obtained about oncogenes, gene regulation, and programmed cell death once again placed Bioenergetics in the limelight of cancer research. Thus, we now have a much better molecular understanding of the high glycolytic phenotype of many cancers, the pivotal roles that Type II hexokinase-mitochondrial interactions play in this process to promote tumor cell growth and survival, and how this new knowledge can lead to improved therapies that may ultimately turn the tide on our losing war on cancer.
Keywords: Cancer; Mitochondria; Glycolysis; Hexokinase; Gene regulation; Drug targeting;
Electron transfer between yeast cytochrome bc 1 complex and cytochrome c: a structural analysis by Carola Hunte; Sozanne Solmaz; Christian Lange (21-28).
The structure of the complex between cytochrome c (CYC) and the cytochrome bc 1 complex (QCR) from yeast crystallized with an antibody fragment has been recently determined at 2.97 Å resolution [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2800]. CYC binds to subunit cytochrome c 1 of the enzyme stabilized by hydrophobic interactions surrounding the heme crevices creating a small, compact contact site. A central cation-π interaction is an important and conserved feature of CYC binding. Peripheral patches with highly conserved complementary charges further stabilize the enzyme–substrate complex by long-range electrostatic forces and may affect the orientation of the substrate. Size and characteristics of the contact site are optimal for a transient electron transfer complex. Kinetic data show a bell-shaped ionic strength dependence of the cytochrome c reduction with a maximum activity near physiological ionic strength. The dependence is less pronounced in yeast compared to horse heart CYC indicating less impact of electrostatic interactions in the yeast system. Interestingly, a local QCR activity minimum is found for both substrates at 120–140 mM ionic strength. The architecture of the complex results in close distance of both c-type heme groups allowing the rapid reduction of cytochrome c by QCR via direct heme-to-heme electron transfer. Remarkably, CYC binds only to one of the two possible binding sites of the homodimeric complex and binding appears to be coordinated with the presence of ubiquinone at the Qi site. Regulatory aspects of CYC reduction are discussed.
Keywords: Ubiquinol:cytochrome c oxidoreductase; Cytochrome c; Electron transfer; Respiration; X-ray crystallography;
Coupling proton movements to c-ring rotation in F1Fo ATP synthase: aqueous access channels and helix rotations at the a–c interface by Robert H Fillingame; Christine M Angevine; Oleg Y Dmitriev (29-36).
F1Fo ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of a ring of c subunits within the transmembrane sector of the enzyme. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Proton access channels to and from aspartyl-61 are thought to form in subunit a of the Fo sector. Here, we summarize new information on the structural organization of subunit a and the mapping of aqueous accessible residues in the fourth and fifth transmembrane helices (TMHs). Cysteine substituted residues, lying on opposite faces of aTMH-4, preferentially react with either N-ethyl-maleimide (NEM) or Ag+. We propose that aTMH-4 rotates to alternately expose each helical face to aspartyl-61 of subunit c during the proton transport cycle. The concerted helical rotation of aTMH-4 and cTMH-2 are proposed to be coupled to the stepwise mechanical movement of the c-rotor.
Keywords: ATP synthase; Proton transport; Rotary motor; Aqueous access channel; Transmembrane helix; Subunit a; Subunit c;
The bc 1 complex of the iron-grown acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans functions in the reverse but not in the forward direction by Gaël Brasseur; Patrice Bruscella; Violaine Bonnefoy; Danielle Lemesle-Meunier (37-43).
Acidithiobacillus ferrooxidans is an acidophilic chemolithotrophic bacterium that can grow in the presence of either a weak reductant, Fe2+, or reducing sulfur compounds that provide more energy for growth than Fe2+. Here we first review the latest findings about the uphill electron transfer pathway established in iron-grown A. ferrooxidans, which has been found to involve a bc 1 complex. We then provide evidence that this bc 1 complex cannot function in the forward direction (exergonic reaction), even with an appropriate substrate. A search for the sequence of the three redox subunits of the A. ferrooxidans bc 1 complex (strain ATCC 19859) in the complete genome sequence of the A. ferrooxidans ATCC 23270 strain showed the existence of two different bc 1 complexes in A. ferrooxidans. Cytochrome b and Rieske protein sequence comparisons allowed us to point out some sequence particularities of these proteins in A. ferrooxidans. Lastly, we discuss the possible reasons for the existence of two different “classical” bc 1 complexes and put forward some suggestions as to what role these putative complexes may play in this acidophilic chemolithotrophic bacterium.
Keywords: Acidithiobacillus ferrooxidans; bc 1 complex; Reverse electron transfer; Cytochrome b; Rieske protein; Acidophilic chemolithotrophic bacteria;
Peroxisomes: surprisingly versatile organelles by Marten Veenhuis; Ida J van der Klei (44-47).
Peroxisome development is a dynamic process that is not yet completely understood. We use the methylotrophic yeast Hansenula polymorpha as model in our studies on peroxisome homeostasis. Cells of this species may contain different types of peroxisomes that differ in protein composition and capacity to incorporate matrix proteins. This protein import machinery is highly flexible and can accommodate unfolded and complex folded proteins.
Keywords: Peroxisome; Organelle; Hansenula polymorpha;
Interactions of quinone with the iron–sulfur protein of the bc 1 complex: is the mechanism spring-loaded? by Antony R Crofts; Vladimir P Shinkarev; Sergei A Dikanov; Rimma I Samoilova; Derrick Kolling (48-53).
Since available structures of native bc 1 complexes show a vacant Qo-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme–substrate complex at the Qo-site. This is formed by binding of both ubiquinol (QH2) and the dissociated oxidized iron–sulfur protein (ISPox). A binding constant of ∼14 can be estimated from the displacement of E m or pK for quinone or ISPox, respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme–product complex is also formed at the Qo-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E m of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E m reflects a binding constant of ∼4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Qo-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.
Keywords: bc 1 complex inhibitor; ESEEM; Ubiquinone-binding; Ubiquinol-binding; Superoxide production; Cytochrome kinetics;
Detection and interpretation of redox potential optima in the catalytic activity of enzymes by Sean J Elliott; Christophe Léger; Harsh R Pershad; Judy Hirst; Kerensa Heffron; Nicolas Ginet; Francis Blasco; Richard A Rothery; Joel H Weiner; Fraser A Armstrong (54-59).
It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation.
Keywords: Oxidoreductase; Voltammetry; Respiration; Electron transport; Redox enzyme; Electrochemistry;
Supramolecular organisation of the photosynthetic chain in anoxygenic bacteria by André Verméglio; Pierre Joliot (60-64).
This minireview summarizes our present view of the supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides and Rhodobacter capsulatus. These two species present a close association between two reaction centers (RCs), one cytochrome (cyt) bc 1 and one cyt c. In R. sphaeroides, the RCs are only partially surrounded by LH1 complexes. This open ring of LH1 complexes is required for an efficient photoinduced cyclic electron transfer only under conditions where the quinone pool totally reduced. When the quinone pool is partially oxidized, a closed ring of LH1 complexes around the RCs does not impair the exchange of quinone molecules between the RC and the cyt bc 1 complex. To explain the efficient photochemistry of the various species which possess a RC surrounded by a closed ring of LH, it is proposed that their quinone pool is partially oxidized even under anaerobic condition.
Keywords: Bacterial photosynthesis; Electron transfer; Supercomplex; Light-harvesting complex; Reaction center;
Inter- and intra-molecular electron transfer in the cytochrome bc 1 complex by Chang-An Yu; Xiaolin Wen; Kunhong Xiao; Di Xia; Linda Yu (65-70).
In this review, we compare the intra-molecular and inter-molecular electron transfer rate constants of the high-potential branch of the cytochrome bc 1 complex. Several methods such as the conventional stopped-flow spectroscopy, pH-induced electron transfer, photoactivated ruthenium complex induced electron transfer and photoreleaseable caged quinol, have been used to determine reaction rates between redox centers in an attempt to elucidate the reaction mechanism of this vital energy conserving complex. Since the most active pure cytochrome bc 1 complex has a turnover number of 800 s−1, any step with a rate constant much larger than this will not be rate-limiting. The most likely rate-limiting step is the cytochrome b redox state governed movement of the head domain of iron–sulfur protein from its electron-accepting site (“fixed” or “b-state” position) to its electron donating site (“c 1-state” position).
Keywords: Cytochrome bc 1 complex; Electron transfer rate; Iron–sulfur protein;
Structure, subunit function and regulation of the coated vesicle and yeast vacuolar (H+)-ATPases by Yoichiro Arata; Tsuyoshi Nishi; Shoko Kawasaki-Nishi; Elim Shao; Stephan Wilkens; Michael Forgac (71-74).
The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function to acidify intracellular compartments in eukaryotic cells. This acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation and the coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in such processes as renal acidification, bone resorption and pH homeostasis. Work from our laboratory has focused on the V-ATPases from clathrin-coated vesicles and yeast vacuoles.Structurally, the V-ATPases are composed of two domains: a peripheral complex (V1) composed of eight different subunits (A–H) that is responsible for ATP hydrolysis and an integral complex (V0) composed of five different subunits (a, d, c, c′ and c″) that is responsible for proton translocation. Electron microscopy has revealed the presence of multiple stalks connecting the V1 and V0 domains, and crosslinking has been used to address the arrangement of subunits in the complex. Site-directed mutagenesis has been employed to identify residues involved in ATP hydrolysis and proton translocation and to study the topology of the 100 kDa a subunit. This subunit has been shown to control intracellular targeting of the V-ATPase and to influence reversible dissociation and coupling of proton transport and ATP hydrolysis.
Keywords: V-ATPase; Proton transport; Membrane protein; Vacuolar acidification; Proton pump structure; Regulation of acidification;
A turbo engine with automatic transmission? How to marry chemicomotion to the subtleties and robustness of life by Sarah Koefoed; Marijke F Otten; Brian J Koebmann; Frank J Bruggeman; Barbara M Bakker; Jacky L Snoep; Klaas Krab; Rob J.M van Spanning; Henk W van Verseveld; Peter R Jensen; Johanna G Koster; Hans V Westerhoff (75-82).
Most genomes are much more complex than required for the minimum chemistry of life. Evolution has selected sophistication more than life itself. Could this also apply to bioenergetics? We first examine mechanisms through which bioenergetics could deliver sophistication. We illustrate possible benefits of the turbo-charging of catabolic pathways, of loose coupling, low-gear catabolism, automatic transmission in energy coupling, and of homeostasis. Mechanisms for such phenomena may reside at the level of individual proton pumps, or consist of rerouting of electrons over parallel pathways. The mechanisms may be confined to preexisting components, or involve the plasticity of gene expression that is so characteristic of most living organisms. These possible benefits lead us to the conjecture that also bioenergetics has evolved more for sophistication than for necessity.We next discuss a hitherto unresolved enigma, i.e. that bioenergetics does not seem to be critical for the physiological state. To decide on how critical bioenergetics is, we quantified the control exerted by catabolism on important physiological functions such as growth rate and growth yield. We also determined whether a growth inhibition mostly affected bioenergetics (catabolism) or anabolism; if ATP increases with growth rate, then growth should be considered energy (catabolism) limited. The experimental results for Escherichia coli pinpoint the enigma: its energy metabolism (catabolism) is not critical for growth rate.These results might suggest that because it has no direct control over cell function, bioenergetics is unimportant. Paradoxically however, in biology, highly important mechanisms tend to have little control on cell function, precisely because of that importance. Sophistication in terms of homeostatic mechanisms has evolved to guarantee robustness of the most important functions: The most important mechanisms are redundant in biology. Bioenergetics may be an excellent example of this paradox, in line with the above conjecture. It may be highly important and sophisticated.We then discuss work that has begun to focus on the sophistication of bioenergetics. Homeostasis of the energetics of DNA structure in E. coli is extensive. It relies both on preexisting components and on responsive gene expression. The vastly parallel electron-transfer network of Paracoccus denitrificans engages in sophisticated dynamic and hierarchical regulation. The growth yield of the organism can depend on which terminal oxidases are active. Effective proton translocation may vary due to rerouting of electrons. We conclude that much sophistication of bioenergetics will be discovered in this era of functional genomics.
Keywords: Hierarchical regulation; Loose coupling; Control; Stoicheiometry; Plasticity; Functional genomics;
Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I by Stefan Kerscher; Stefan Dröse; Klaus Zwicker; Volker Zickermann; Ulrich Brandt (83-91).
The obligate aerobic yeast Yarrowia lipolytica is introduced as a powerful new model for the structural and functional analysis of mitochondrial complex I. A brief introduction into the biology and the genetics of this nonconventional yeast is given and the relevant genetic tools that have been developed in recent years are summarized. The respiratory chain of Y. lipolytica contains complexes I–IV, one “alternative” NADH-dehydrogenase (NDH2) and a non-heme alternative oxidase (AOX). Because the NADH binding site of NDH2 faces the mitochondrial intermembrane space rather than the matrix, complex I is an essential enzyme in Y. lipolytica. Nevertheless, complex I deletion strains could be generated by attaching the targeting sequence of a matrix protein, thereby redirecting NDH2 to the matrix side. Deletion strains for several complex I subunits have been constructed that can be complemented by shuttle plasmids carrying the deleted gene. Attachment of a hexa-histidine tag to the NUGM (30 kDa) subunit allows fast and efficient purification of complex I from Y. lipolytica by affinity-chromatography. The purified complex has lost most of its NADH:ubiquinone oxidoreductase activity, but is almost fully reactivated by adding 400–500 molecules of phosphatidylcholine per complex I. The established set of genetic tools has proven useful for the site-directed mutagenesis of individual subunits of Y. lipolytica complex I. Characterization of a number of mutations already allowed for the identification of several functionally important amino acids, demonstrating the usefulness of this approach.
Keywords: Yarrowia lipolytica; Complex I; NADH:ubiquinone oxidoreductase; Alternative NADH dehydrogenase; Alternative oxidase;
The dual-function glutamate transporters: structure and molecular characterisation of the substrate-binding sites by B.I Kanner; L Borre (92-95).
Glutamate transporters are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentrations below neurotoxic levels. These transporters also mediate a thermodynamically uncoupled chloride flux, activated by two of the molecules they transport, sodium and glutamate. Five eukaryotic glutamate transporters have been cloned and identified. They exhibit ∼50% identity and this homology is even greater at the carboxyl terminal half, which is predicted to have an unusual topology. Determination of the topology shows that the carboxyl terminal part contains several transmembrane domains separated by two reentrant loops that are in close proximity to each other. We have identified several conserved amino acid residues in the carboxyl terminal half that play crucial roles in the interaction of the transporter with its substrates: sodium, potassium and glutamate. The conformation of the transporter gating the anion conductance is different from that during substrate translocation. However, there exists a dynamic equilibrium between these conformations.
Keywords: Sodium and potassium coupling; Anion conductance; Topology; Proximity relation; Sequential binding; Dynamic equilibrium;
Influence of structure, pH and membrane potential on proton movement in cytochrome oxidase by Denise A Mills; Shelagh Ferguson-Miller (96-100).
Cytochrome c oxidase (CcO) reconstituted into phospholipid vesicles and subject to a membrane potential, exhibits different characteristics than the free enzyme, with respect to effects of mutations, pH, inhibitors, and native structural differences between CcO from different species. The results indicate that the membrane potential influences the conformation of CcO and the direction of proton movement in the exit path. The importance of the protein structure above the hemes in proton exit, back leak and respiratory control is discussed.
Keywords: Cytochrome oxidase; Proton pathway; Respiratory control; Proton exit;
Atp11p and Atp12p are chaperones for F1-ATPase biogenesis in mitochondria by Sharon H Ackerman (101-105).
The bioenergetic needs of aerobic cells are met principally through the action of the F1F0 ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F1) is a multimeric protein of the subunit composition α3β3γδε. Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial α and β subunits into the F1 oligomer requires two molecular chaperone proteins called Atp11p and Atp12p. Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells.
Keywords: Mitochondria; F1-ATPase; Atp11p; Atp12p; Chaperone; Biogenesis;
Manipulation of mitochondrial DNA gene expression in the mouse by José P Silva; Nils-Göran Larsson (106-110).
Mitochondrial dysfunction due to impaired respiratory chain function is increasingly recognized as an important cause of human disease. Mitochondrial disorders are relatively common and have an estimated incidence of 1:10,000 live births. There are more than 100 different point mutations and numerous large rearrangements of mitochondrial DNA (mtDNA; mainly single deletions) that cause human disease. We aimed at obtaining an animal model to study physiological aspects of mtDNA mutation disorders. There are as yet unsolved technical problems associated with transfection of mammalian mitochondria. We therefore choose to manipulate mtDNA expression by targeting of the nuclear gene encoding Tfam. We utilised the cre-loxP recombination system to disrupt Tfam since this system allows manipulation of respiratory chain function in selected mouse tissues. We have found increased cell death or apoptosis induction in both germ line and tissue-specific Tfam knockouts. Our results further suggest that increased production of reactive oxygen species (ROS) is not a prominent feature in cells with impaired mtDNA expression.
Keywords: Apoptosis; Aging; mtDNA mutation; ROS; Mouse model; cre-loxP;
Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry by Bart A van Montfort; Mark K Doeven; Benito Canas; Liesbeth M Veenhoff; Bert Poolman; George T Robillard (111-115).
A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.
Keywords: Mass spectrometry; Peptide mapping; Membrane protein; Matrix-assisted laser desorption/ionization; CNBr; trypsin;
ATR-FTIR difference spectroscopy of the PM intermediate of bovine cytochrome c oxidase by Masayo Iwaki; Jacques Breton; Peter R Rich (116-121).
Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic PM intermediate. The transition was induced in a film of detergent-depleted ‘fast’ oxidase with a buffer containing CO and O2. The extent of formation of the PM state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The PM minus O FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a 3 and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds.
Keywords: Cytochrome c oxidase; FTIR spectroscopy; Intermediate;
The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli by Tania Bizouarn; Magnus Althage; Anders Pedersen; Anna Tigerström; Jenny Karlsson; Carina Johansson; Jan Rydström (122-127).
Proton-translocating nicotinamide nucleotide transhydrogenase is a conformationally driven pump which catalyzes the reversibel reduction of NADP+ by NADH. Transhydrogenases contain three domains, i.e., the hydrophilic NAD(H)-binding domain I and the NADP(H)-binding domain III, and the hydrophobic domain II containing the proton channel. Domains I and III have been separately expressed and characterized structurally by, e.g. X-ray crystallography and NMR. These domains catalyze transhydrogenation in the absence of domain II. However, due to the absence of the latter domain, the reactions catalyzed by domains I and III differ significantly from those catalyzed by the intact enzyme. Mutagenesis of residues in domain II markedly affects the activity of the intact enzyme. In order to resolve the structure–function relationships of the intact enzyme, and the molecular mechanism of proton translocation, it is therefore essential to establish the structure and function of domain II and its interactions with domains I and III. This review describes some relevant recent results in this field of research.
Keywords: Transhydrogenase; NADP; NAD; Membrane protein; Proton pump; Proton translocation;
Proton translocation by cytochrome c oxidase in different phases of the catalytic cycle by Mårten Wikström; Michael I Verkhovsky (128-132).
Since mitochondrial cytochrome c oxidase was found to be a redox-linked proton pump, most enzymes of the haem-copper oxidase family have been shown to share this function. Here, the most recent knowledge of how the individual reactions of the enzyme's catalytic cycle are coupled to proton translocation is reviewed. Two protons each are pumped during the oxidative and reductive halves of the cycle, respectively. An apparent controversy that concerns proton translocation during the reductive half is resolved. If the oxidised enzyme is allowed to relax in the absence of reductant, the binuclear haem-copper centre attains a state that lies outside the main catalytic cycle. Reduction of this form of the enzyme is not linked to proton translocation, but is necessary for a return to the main cycle. This phenomenon might be related to the previously described “pulsed” vs. “resting” and “fast” vs.“slow” forms of haem-copper oxidases.
Keywords: Proton translocation; Cytochrome c oxidase; Catalytic cycle;
Inhibition of proton transfer in cytochrome c oxidase by zinc ions: delayed proton uptake during oxygen reduction by Anna Aagaard; Andreas Namslauer; Peter Brzezinski (133-139).
We have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn2+ the transition from the “peroxy” (PR) to the “ferryl” (F) intermediate has a time constant of ∼100 μs and it is associated with proton transfer from the bulk solution with an intrinsic time constant of ≪100 μs, but rate limited by the PR→F transition. While in the presence of 100 μM Zn2+ the PR→F transition was slowed by a factor of ∼2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn2+) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of ∼2.5 ms. Thus, the results show that there is one proton available within the enzyme that can be used for oxygen reduction and confirm our previous observation that F can be formed without proton uptake from the bulk solution. No effect of Zn2+ was observed with a mutant enzyme in which Asp(I-132), at the entry point of the D-pathway, was replaced by its non-protonatable analogue Asn. In addition, no effect of Zn2+ was observed on the F→O transition rate when measured in D2O, because in D2O, the transition is internally slowed to ∼10 ms, which is already slower than with bound Zn2+. Together with earlier results showing that both the PR→F and F→O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn2+ binds to and blocks the entrance of the D-pathway.
Keywords: Electron transfer; Flow-flash; Proton pumping; Cytochrome aa 3; Flash photolysis; Rhodobacter sphaeroides;
Molecular devices of chloroplast F1-ATP synthase for the regulation by Toru Hisabori; Hiroki Konno; Hiroki Ichimura; Heinrich Strotmann; Dirk Bald (140-146).
In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (ΔμH+) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems ΔμH+ activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit ε is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level.
Keywords: Chloroplast; ATP synthase; Redox regulation; γ subunit; ε subunit;
The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade by Sergio Papa (147-153).
Results of studies on the role of the 18 kDa (IP) polypeptide subunit of complex I, encoded by the nuclear NDUFS4 gene, in isolated bovine heart mitochondria and human and murine cell cultures are presented.The mammalian 18 kDa subunit has in the carboxy-terminal sequence a conserved consensus site (RVS), which in isolated mitochondria is phosphorylated by cAMP-dependent protein kinase (PKA). The catalytic and regulatory subunits of PKA have been directly immunodetected in the inner membrane/matrix fraction of mammalian mitochondria. In the mitochondrial inner membrane a PP2Cγ-type phosphatase has also been immunodetected, which dephosphorylates the 18 kDa subunit, phosphorylated by PKA. This phosphatase is Mg2+-dependent and inhibited by Ca2+. In human and murine fibroblast and myoblast cultures “in vivo”, elevation of intracellular cAMP level promotes phosphorylation of the 18 kDa subunit and stimulates the activity of complex I and NAD-linked mitochondrial respiration.Four families have been found with different mutations in the cDNA of the NDUFS4 gene. These mutations, transmitted by autosomal recessive inheritance, were associated in homozygous children with fatal neurological syndrome. All these mutations destroyed the phosphorylation consensus site in the C terminus of the 18 kDa subunit, abolished cAMP activation of complex I and impaired its normal assembly.
Keywords: Mitochondrion; Complex I; NDUFS4 gene; cAMP-cascade;
Respiratory chain supercomplexes of mitochondria and bacteria by Hermann Schägger (154-159).
Respiratory chain complexes are fragments of larger structural and functional units, the respiratory chain supercomplexes or “respirasomes“, which exist in bacterial and mitochondrial membranes. Supercomplexes of mitochondria and bacteria contain complexes III, IV, and complex I, with the notable exception of Saccharomyces cerevisiae, which does not possess complex I. These supercomplexes often are stable to sonication but sensitive to most detergents except digitonin. In S. cerevisiae, a major component linking complexes III and IV together is cardiolipin.In Paracoccus denitrificans, complex I itself is rather detergent-sensitive and thus could not be obtained in detergent-solubilized form so far. However, it can be isolated as part of a supercomplex. Stabilization of complex I by binding to complex III was also found in human mitochondria. Further functional roles of the organization in a supercomplex are catalytic enhancement by reducing diffusion distances of substrates or, depending on the organism, channelling of the substrates quinone and cytochrome c. This makes redox reactions less dependent of midpoint potentials of substrates, and permits electron flow at low degree of substrate reduction.A dimeric state of ATP synthase seems to be specific for mitochondria. Exclusively, monomeric ATP synthase was found in Acetobacterium woodii, in P. denitrificans, and in spinach chloroplasts.
Keywords: Respiratory chain; Supercomplex; Oxidative phosphorylation; Mitochondria; Paracoccus denitrificans; Saccharomyces cerevisiae;
Effects of fatty acids on mitochondria: implications for cell death by Daniele Penzo; Chiara Tagliapietra; Raffaele Colonna; Valeria Petronilli; Paolo Bernardi (160-165).
Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.
Keywords: Fatty acids; Mitochondria; Membrane potential; Cell death;
A concerted, alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc 1 complex by Bernard L Trumpower (166-173).
A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske iron–sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c 1. A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol–cytochrome c reductase kinetics of the bc 1 complex include both a pH dependence and a dependence on Rieske iron–sulfur protein midpoint potential.An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc 1 complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc 1 complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound.An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc 1 activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.
Keywords: Cytochrome bc 1 complex; Q cycle; Ubiquinol; Rieske iron–sulfur protein; Concerted mechanism;
Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? by Marie-France Giraud; Patrick Paumard; Vincent Soubannier; Jacques Vaillier; Geneviève Arselin; Bénédicte Salin; Jacques Schaeffer; Daniel Brèthes; Jean-Paul di Rago; Jean Velours (174-180).
Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers. Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane. We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases. Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane. From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers. We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis.
Keywords: ATP synthase; Cristae; Morphology; Dimerization; F0 subunit; Mitochondria; Yeast;
Aquaglyceroporins, one channel for two molecules by Daniel Thomas; Patrick Bron; Grégory Ranchy; Laurence Duchesne; Annie Cavalier; Jean-Paul Rolland; Céline Raguénès-Nicol; Jean-François Hubert; Winfried Haase; Christian Delamarche (181-186).
In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane.
Keywords: Aquaporin; Glycerol facilitator; Aquaglyceroporin; Selectivity filter; Sequence analysis; Freeze-fracture;
From NADH to ubiquinone in Neurospora mitochondria by Arnaldo Videira; Margarida Duarte (187-191).
The respiratory chain of the mitochondrial inner membrane includes a proton-pumping enzyme, complex I, which catalyses electron transfer from NADH to ubiquinone. This electron pathway occurs through a series of protein-bound prosthetic groups, FMN and around eight iron–sulfur clusters. The high number of polypeptide subunits of mitochondrial complex I, around 40, have a dual genetic origin. Neurospora crassa has been a useful genetic model to characterise complex I. The characterisation of mutants in specific proteins helped to understand the elaborate processes of the biogenesis, structure and function of the oligomeric enzyme. In the fungus, complex I seems to be dispensable for vegetative growth but required for sexual development. N. crassa mitochondria also contain three to four nonproton-pumping alternative NAD(P)H dehydrogenases. One of them is located in the outer face of the inner mitochondrial membrane, working as a calcium-dependent oxidase of cytosolic NADPH.
Keywords: Mitochondria; Respiratory chain; Complex I; Alternative NADH dehydrogenase; Mutant; Neurospora crassa;
A replicating module as the unit of mitochondrial structure and functioning by Roderick A Capaldi; Robert Aggeler; Robert Gilkerson; George Hanson; Michelle Knowles; Andrew Marcus; Daciana Margineantu; Michael Marusich; James Murray; Devin Oglesbee; S.James Remington; Rodrigue Rossignol (192-195).
The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in ≈0.8-μm jumps that are determined by, and related to, attachments with cytoskeletal elements. Many protein complexes, such as the pyruvate dehydrogenase (PDH) complex and DNA containing nucleoids, are dispersed through the mass and as though fixed by attachments to membranes, such that they can become distributed to all of the individual small ovoid mitochondria when the reticulum becomes fragmented. This leads us to propose that a replicating module is the repeating unit of mitochondrial structure. Studies to examine heterogeneity of functioning within the organelle mass are briefly reviewed.
Keywords: Replicating module; Mitochondrion; Structure–function relationship;
Insight into mitochondrial structure and function from electron tomography by T.G Frey; C.W Renken; G.A Perkins (196-203).
In recent years, electron tomography has provided detailed three-dimensional models of mitochondria that have redefined our concept of mitochondrial structure. The models reveal an inner membrane consisting of two components, the inner boundary membrane (IBM) closely apposed to the outer membrane and the cristae membrane that projects into the matrix compartment. These two components are connected by tubular structures of relatively uniform size called crista junctions. The distribution of crista junction sizes and shapes is predicted by a thermodynamic model based upon the energy of membrane bending, but proteins likely also play a role in determining the conformation of the inner membrane. Results of structural studies of mitochondria during apoptosis demonstrate that cytochrome c is released without detectable disruption of the outer membrane or extensive swelling of the mitochondrial matrix, suggesting the formation of an outer membrane pore large enough to allow passage of holo-cytochrome c. The possible compartmentation of inner membrane function between the IBM and the cristae membrane is also discussed.
Keywords: Electron microscopy; Electron tomography; Mitochondria; Membrane topology; Three-dimensional imaging; Bioenergetics;
The ferredoxin docking site of photosystem I by Pierre Sétif; Nicolas Fischer; Bernard Lagoutte; Hervé Bottin; Jean-David Rochaix (204-209).
The reaction center of photosystem I (PSI) reduces soluble ferredoxin on the stromal side of the photosynthetic membranes of cyanobacteria and chloroplasts. The X-ray structure of PSI from the cyanobacterium Synechococcus elongatus has been recently established at a 2.5 Å resolution [Nature 411 (2001) 909]. The kinetics of ferredoxin photoreduction has been studied in recent years in many mutants of the stromal subunits PsaC, PsaD and PsaE of PSI. We discuss the ferredoxin docking site of PSI using the X-ray structure and the effects brought by the PSI mutations to the ferredoxin affinity.
Keywords: Photosynthesis; Mutagenesis; Electrostatic interaction; Membrane protein complex; Electron transfer; Iron–sulfur center;
Bioenergetics Author Index (211-213).
Bioenergetics Cumulative Contents (215-216).