BBA - Bioenergetics (v.1459, #2-3)
Revealing the structure of the photosystem II chlorophyll binding proteins, CP43 and CP47 by J Barber; E Morris; C Büchel (239-247).
A review of the structural properties of the photosystem II chlorophyll binding proteins, CP47 and CP43, is given and a model of the transmembrane helical domains of CP47 has been constructed. The model is based on (i) the amino acid sequence of the spinach protein, (ii) an 8 Å three-dimensional electron density map derived from electron crystallography and (iii) the structural homology which the membrane spanning region of CP47 shares with the six N-terminal transmembrane helices of the PsaA/PsaB proteins of photosystem I. Particular emphasis has been placed on the position of chlorophyll molecules assigned in the 8 Å three-dimensional map of CP47 (K.-H. Rhee, E.P. Morris, J. Barber, W. Kühlbrandt, Nature 396 (1998) 283–286) relative to histidine residues located in the transmembrane regions of this protein which are likely to form axial ligands for chlorophyll binding. Of the 14 densities assigned to chlorophyll, the model predicted that five have their magnesium ions within 4 Å of the imidazole nitrogens of histidine residues. For the remaining seven histidine residues the densities attributed to chlorophylls were within 4–8 Å of the imidazole nitrogens and thus too far apart for direct ligation with the magnesium ion within the tetrapyrrole head group. Improved structural resolution and reconsiderations of the orientation of the porphyrin rings will allow further refinement of the model.
Keywords: Photosynthesis; Photosystem II; Structure; CP47; CP43; Chlorophyll binding protein;
Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum by Mark Jeeves; K.John Smith; Philip G Quirk; Nick P.J Cotton; J.Baz Jackson (248-257).
Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria. The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods. This is the first description of the structure of dIII from a bacterial source. The protein adopts a Rossmann fold: an open, twisted, parallel β-sheet, flanked by helices. However, the binding of NADP+ to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first βαβαβ motif, and the nicotinamide with the second. Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified. The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.
Keywords: Transhydrogenase; Membrane protein; Proton translocation; Nuclear magnetic resonance structure; Nucleotide binding;
Application of the obligate aerobic yeast Yarrowia lipolytica as a eucaryotic model to analyse Leigh syndrome mutations in the complex I core subunits PSST and TYKY by Pamela M Ahlers; Aurelio Garofano; Stefan J Kerscher; Ulrich Brandt (258-265).
We have used the obligate aerobic yeast Yarrowia lipolytica to reconstruct and analyse three missense mutations in the nuclear coded subunits homologous to bovine TYKY and PSST of mitochondrial complex I (proton translocating NADH:ubiquinone oxidoreductase) that have been shown to cause Leigh syndrome (MIM 25600), a severe progressive neurodegenerative disorder. While homozygosity for a V122M substitution in NDUFS7 (PSST) has been found in two siblings with neuropathologically proven Leigh syndrome (R. Triepels et al., Ann. Neurol. 45 (1999) 787), heterozygosity for a P79L and a R102H substitution in NDUFS8 (TYKY) has been found in another patient (J. Loeffen et al., Am. J. Hum. Genet. 63 (1998) 1598). Mitochondrial membranes from Y. lipolytica strains carrying any of the three point mutations exhibited similar complex I defects, with V max being reduced by about 50%. This suggests that complex I mutations that clinically present as Leigh syndrome may share common characteristics. In addition changes in the K m for n-decyl-ubiquinone and I 50 for hydrophobic complex I inhibitors were observed, which provides further evidence that not only the hydrophobic, mitochondrially coded subunits, but also some of the nuclear coded subunits of complex I are involved in its reaction with ubiquinone.
Keywords: Mitochondria; Complex I; Yeast; Yarrowia lipolytica; Leigh syndrome; Point mutation;
Nitric oxide reductases in bacteria by Janneke Hendriks; Arthur Oubrie; Jose Castresana; Andrea Urbani; Sabine Gemeinhardt; Matti Saraste (266-273).
Nitric oxide reductases (NORs) that are found in bacteria belong to the large enzyme family which includes cytochrome oxidases. Two types of bacterial NORs have been characterised. One is a cytochrome bc-type complex (cNOR) that receives electrons from soluble redox protein donors, whereas the other type (qNOR) lacks the cytochrome c component and uses quinol as the electron donor. The latter enzyme is present in several pathogens that are not denitrifiers. We summarise the current knowledge on bacterial NORs, and discuss the evolutionary relationship between them and cytochrome oxidases in this review.
Keywords: Denitrification; Bacterium; Nitric oxide reductase; Evolution;
Diversity and origin of alternative NADH:ubiquinone oxidoreductases by Stefan J Kerscher (274-283).
Mitochondria from various organisms, especially plants, fungi and many bacteria contain so-called alternative NADH:ubiquinone oxidoreductases that catalyse the same redox reaction as respiratory chain complex I, but do not contribute to the generation of transmembrane proton gradients. In eucaryotes, these enzymes are associated with the mitochondrial inner membrane, with their NADH reaction site facing either the mitochondrial matrix (internal alternative NADH:ubiquinone oxidoreductases) or the cytoplasm (external alternative NADH:ubiquinone oxidoreductases). Some of these enzymes also accept NADPH as substrate, some require calcium for activity. In the past few years, the characterisation of several alternative NADH:ubiquinone oxidoreductases on the DNA and on the protein level, of substrate specificities, mitochondrial import and targeting to the mitochondrial inner membrane has greatly improved our understanding of these enzymes. The present review will, with an emphasis on yeast model systems, illuminate various aspects of the biochemistry of alternative NADH:ubiquinone oxidoreductases, address recent developments and discuss some of the questions still open in the field.
Keywords: Alternative NADH:ubiquinone oxidoreductase; Alternative NADH dehydrogenase; Respiratory chain; Electron transport;
The transmembrane domain and the proton channel in proton-pumping transhydrogenases by Tania Bizouarn; Johan Meuller; Magnus Axelsson; Jan Rydström (284-290).
Proton-pumping nicotinamide nucleotide transhydrogenases are composed of three main domains, the NAD(H)-binding and NADP(H)-binding hydrophilic domains I (dI) and III (dIII), respectively, and the hydrophobic domain II (dII) containing the assumed proton channel. dII in the Escherichia coli enzyme has recently been characterised with regard to topology and a packing model of the helix bundle in dII is proposed. Extensive mutagenesis of conserved charged residues of this domain showed that important residues are βHis91 and βAsn222. The pH dependence of βH91D, as well as βH91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD+ by NADPH, i.e., the reverse reaction, is optimal at a pH essentially coinciding with the pK a of the residue in the β91 position. It is therefore concluded that the wild-type transhydrogenase is regulated by the degree of protonation of βHis91. The mechanisms of the interactions between dI+dIII and dII are suggested to involve pronounced conformational changes in a ‘hinge’ region around βR265.
Keywords: Transhydrogenase; Proton pump; Membrane protein; Nicotinamide nucleotide; Nicotinamide adenine dinucleotide; Nicotinamide adenine dinucleotide phosphate;
Mitochondrial electron transfer in the wheat pathogenic fungus Septoria tritici: on the role of alternative respiratory enzymes in fungicide resistance by Charles Affourtit; Steve P. Heaney; Anthony L. Moore (291-298).
Certain phytopathogenic fungi are able to express alternative NADH- and quinol-oxidising enzymes that are insensitive to inhibitors of the mitochondrial respiratory Complexes I and III. To assess the extent to which such enzymes confer tolerance to respiration-targeted fungicides, an understanding of mitochondrial electron transfer in these species is required. An isolation procedure has been developed which results in intact, active and coupled mitochondria from the wheat pathogen Septoria tritici, as evidenced by morphological and kinetic data. Exogenous NADH, succinate and malate/glutamate are readily oxidised, the latter activity being only partly (approx. 70%) sensitive to rotenone. Of particular importance was the finding that azoxystrobin (a strobilurin fungicide) potently inhibits fungal respiration at the level of Complex III. In some S. tritici strains investigated, a small but significant part of the respiratory activity (approx. 10%) is insensitive to antimycin A and azoxystrobin. Such resistant activity is sensitive to octyl gallate, a specific inhibitor of the plant alternative oxidase. This enzyme, however, could not be detected immunologically. On the basis of the above findings, a conceptual mitochondrial electron transfer chain is presented. Data are discussed in terms of developmental and environmental regulation of the composition of this chain.
Keywords: Phytopathogen; Respiration-targeted fungicide; Mitochondrial isolation; Electron transfer chain; Mycosphaerella graminicola; Azoxystrobin;
Characterization of the complex I-associated ubisemiquinone species: toward the understanding of their functional roles in the electron/proton transfer reaction by Takahiro Yano; Sergey Magnitsky; Tomoko Ohnishi (299-304).
NADH-ubiquinone oxidoreductase (called complex I for mitochondrial enzyme and NDH-1 for bacterial counterparts) is an energy transducer, which utilizes the redox energy derived from the oxidation of NADH with ubiquinone to generate an electrochemical proton gradient (Δ μ ̃ H + ) across the membrane. The complex I/NDH-1 contain one non-covalently bound flavin mononucleotide and as many as eight iron-sulfur clusters as electron transfer components in common. In addition, electron paramagnetic resonance (EPR) spectroscopic studies have revealed that three ubisemiquinone (SQ) species with distinct spectroscopic and thermodynamic properties are detectable in complex I and function as electron/proton translocators. Thus, the understanding of molecular properties of the individual quinone species is prerequisite to elucidate the energy-coupling mechanism of complex I. We have investigated these SQ species using EPR spectroscopy and found that the three SQ species have strikingly different properties. We will report characteristics of these SQ species and discuss possible functional roles of individual quinone species in the electron/proton transfer reaction of complex I/NDH-1.
Keywords: NADH-ubiquinone oxidoreductase; Complex I; Electron paramagnetic resonance; Energy transduction; Ubisemiquinone; Iron-sulfur cluster;
Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I) by Thorsten Friedrich; Benedikt Brors; Petra Hellwig; Lars Kintscher; Tim Rasmussen; Dierk Scheide; Ulrich Schulte; Werner Mäntele; Hanns Weiss (305-309).
The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 α-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of −150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of −80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Fourier transformed infrared spectroscopy; Electron paramagnetic resonance spectroscopy; Escherichia coli; Neurospora crassa;
Catalysis in fumarate reductase by Graeme A. Reid; Caroline S. Miles; Ruth K. Moysey; Katherine L. Pankhurst; Stephen K. Chapman (310-315).
In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c 3 that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.
Keywords: Bacterial respiration; Fumarate reductase; Shewanella; Flavoprotein;
Haem-polypeptide interactions during cytochrome c maturation by Linda Thöny-Meyer (316-324).
Cytochrome c maturation involves the translocation of a polypeptide, the apocytochrome, and its cofactor, haem, through a membrane, before the two molecules are ligated covalently. This review article focusses on the current knowledge on the journey of haem during this process, which is known best in the Gram-negative bacterium Escherichia coli. As haem always occurs bound to protein, its passage across the cytoplasmic membrane and incorporation into the apocytochrome appears to be mediated by a set of proteinaceous maturation factors, the Ccm ( c ytochrome c m aturation) proteins. At least three of them, CcmC, CcmE and CcmF, are thought to interact directly with haem. CcmE binds haem covalently, thus representing an intermediate of the haem trafficking pathway. CcmC is required for binding of haem to CcmE, and CcmF for releasing it from CcmE and transferring it onto the apocytochrome. The mechanism by which haem crosses the cytoplasmic membrane is currently unknown.
Keywords: ccm gene; Cofactor; Cytochrome c biogenesis; Haem trafficking; Membrane protein complex;
A novel protein transport system involved in the biogenesis of bacterial electron transfer chains by Ben C. Berks; Frank Sargent; Erik De Leeuw; Andrew P. Hinsley; Nicola R. Stanley; Rachael L. Jack; Grant Buchanan; Tracy Palmer (325-330).
The Tat system is a recently discovered bacterial protein transport pathway that functions primarily in the biosynthesis of proteins containing redox active cofactors. Analogous transport systems are found in plant organelles. Remarkably and uniquely the Tat system functions to transported a diverse range of folded proteins across a biological membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions. Here we review the operation of the bacterial Tat system and propose a model for the structural organisation of the Tat preprotein translocase.
Keywords: Protein transport; Redox protein; Metalloprotein biosynthesis; Tat pathway; Escherichia coli;
What fuels polypeptide translocation? An energetical view on mitochondrial protein sorting by Johannes M. Herrmann; Walter Neupert (331-338).
Protein sorting into mitochondria is achieved by the concerted action of at least four translocation complexes. Vectorial transport of polypeptide chains by these complexes requires different driving forces. In particular, Δψ, matrix adenosine triphosphate and the free energy of the binding to other protein components are used in series to achieve sorting of proteins to the various mitochondrial subcompartments. The processes providing the translocation energy are presented in this review and their impact for protein sorting into and within mitochondria is discussed.
Keywords: Mitochondria; Protein translocation; Translocase; Brownian ratchet; Membrane potential; Adenosine triphosphate; Driving force;
Crystallographic studies of the conformational changes that drive directional transmembrane ion movement in bacteriorhodopsin by Janos K Lanyi (339-345).
Recent advances in the determination of the X-ray crystallographic structures of bacteriorhodopsin, and some of its photointermediates, reveal the nature of the linkage between the relaxation of electrostatic and steric conflicts at the retinal and events elsewhere in the protein. The transport cycle can be now understood in terms of specific and well-described displacements of hydrogen-bonded water, and main-chain and side-chain atoms, that lower the pK as of the proton release group in the extracellular region and Asp-96 in the cytoplasmic region. Thus, local electrostatic conflict of the photoisomerized retinal with Asp-85 and Asp-212 causes deprotonation of the Schiff base, and results in a cascade of events culminating in proton release to the extracellular surface. Local steric conflict of the 13-methyl group with Trp-182 causes, in turn, a cascade of movements in the cytoplasmic region, and results in reprotonation of the Schiff base. Although numerous questions concerning the mechanism of each of these proton (or perhaps hydroxyl ion) transfers remain, the structural results provide a detailed molecular explanation for how the directionality of the ion transfers is determined by the configurational relaxation of the retinal.
Keywords: Bacteriorhodopsin; Retinal; Proton transport; Protein structure;
Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea by Mark Whittaker; David Bergmann; David Arciero; Alan B Hooper (346-355).
The combined action of ammonia monooxygenase, AMO, (NH3+2e−+O2→NH2OH) and hydroxylamine oxidoreductase, HAO, (NH2OH+H2O→HNO2+4e−+4H+) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O2, nitrite, NO, H2O2 or AMO are reviewed and some recent advances described. The membrane cytochrome c M552 is proposed to participate in the path between HAO and ubiquinone. A bc 1 complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N2O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O2; the flux of nitrite reduction is controlled at the substrate level.
Mammalian mitochondrial inner membrane cationic and neutral amino acid carriers by Richard K Porter (356-362).
Keywords: Mitochondria; Transporter; Neutral; Glycine; Proline; Glutamine; Arginine; Carnitine; Ornithine; Citrulline; Choline;
Identification and functions of new transporters in yeast mitochondria by Luigi Palmieri; Francesco M. Lasorsa; Angelo Vozza; Gennaro Agrimi; Giuseppe Fiermonte; Michael J. Runswick; John E. Walker; Ferdinando Palmieri (363-369).
The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.
Keywords: Yeast; Mitochondria; Transport; Gene; Overexpression; Carrier; Metabolism;
Biogenesis of iron–sulfur proteins in eukaryotes: a novel task of mitochondria that is inherited from bacteria by Ulrich Mühlenhoff; Roland Lill (370-382).
Fe/S clusters are co-factors of numerous proteins with important functions in metabolism, electron transport and regulation of gene expression. Presumably, Fe/S proteins have occurred early in evolution and are present in cells of virtually all species. Biosynthesis of these proteins is a complex process involving numerous components. In mitochondria, this process is accomplished by the so-called ISC ( i ron– s ulfur c luster assembly) machinery which is derived from the bacterial ancestor of the organelles and is conserved from lower to higher eukaryotes. The mitochondrial ISC machinery is responsible for biogenesis iron–sulfur proteins both within and outside the organelle. Maturation of the latter proteins involves the ABC transporter Atm1p which presumably exports iron–sulfur clusters from the organelle. This review summarizes recent developments in our understanding of the biogenesis of iron–sulfur proteins both within bacteria and eukaryotes.
How do uncoupling proteins uncouple? by Keith D. Garlid; Martin Jabůrek; Petr Ježek; Miroslav Vařecha (383-389).
According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.
Keywords: Mitochondria; Uncoupling protein; Fatty acid; Nucleotide; Ion transport; Reconstitution;
Effects of nitric oxide and peroxynitrite on the cytochrome oxidase K m for oxygen: implications for mitochondrial pathology by Chris E Cooper; Nathan A Davies (390-396).
This review summarises current knowledge about the effect of oxygen on cytochrome oxidase activity in vitro and in vivo. Cytochrome oxidase normally operates above its K m for oxygen in vivo. However, decreases in the intracellular oxygen concentration (hypoxia) under physiological extremes, or during pathophysiology, can cause mitochondrial respiration to become oxygen limited. Inhibitors that raise the enzyme’s K m will induce oxygen limitation under apparently normoxic conditions. It is known that the concentrations of nitric oxide and peroxynitrite are raised in a number of pathophysiological conditions. These compounds are capable of reversibly and irreversibly raising the cytochrome oxidase K m for oxygen. Therefore, measurements of cell and mitochondrial respiration in vitro that fail to systematically vary oxygen through the range of physiological concentrations are likely to underestimate the effects of nitric oxide and peroxynitrite in vivo.
Keywords: Nitric oxide; Peroxynitrite; Cytochrome oxidase; Oxygen; K m; Mitochondria;
Mitochondrial bioenergetics in aging by G Lenaz; M D’Aurelio; M Merlo Pich; M.L Genova; B Ventura; C Bovina; G Formiggini; G Parenti Castelli (397-404).
Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.
Keywords: Mitochondria; Aging; Respiratory chain; Coenzyme Q; Metabolic control; Platelet;
Reversal of nitric oxide-, peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols by Vilmante Borutaite; Aiste Budriunaite; Guy C. Brown (405-412).
Nitric oxide (NO) and its derivatives peroxynitrite and S-nitrosothiols inhibit mitochondrial respiration by various means, but the mechanisms and/or the reversibility of such inhibitions are not clear. We find that the NO-induced inhibition of respiration in isolated mitochondria due to inhibition of cytochrome oxidase is acutely reversible by light. Light also acutely reversed the inhibition of respiration within iNOS-expressing macrophages, and this reversal was partly due to light-induced breakdown of NO, and partly due to reversal of the NO-induced inhibition of cytochrome oxidase. NO did not cause inhibition of complex I activity within isolated mitochondria, but 0.34 mM peroxynitrite, 1 mM S-nitroso-N-acetylpenicillamine or 1 mM S-nitrosoglutathione did cause substantial inhibition of complex I activity. Inhibition by these reagents was reversed by light, dithiothreitol or glutathione-ethyl ester, either partially or completely, depending on the reagent used. The rapid inhibition of complex I activity by S-nitroso-N-acetylpenicillamine also occurred in conditions where there was little or no release of free NO, suggesting that the inhibition was due to transnitrosylation of the complex. These findings have implications for the physiological and pathological regulation of respiration by NO and its derivatives.
Keywords: Nitric oxide; Mitochondria; Cytochrome oxidase; Respiration; Oxygen consumption;
Re-emerging structures: continuing crystallography of the bacterial reaction centre by Paul K Fyfe; Michael R Jones (413-421).
The reaction centre is nature’s solar battery, and is found in a number of variations on a common theme in plants, algae and photosynthetic bacteria. During the last 20 years, a combination of X-ray crystallography, spectroscopy and mutagenesis has provided increasingly detailed insights into the mechanism of light energy transduction in the bacterial reaction centre. This mini-review looks at the application of X-ray crystallography to the bacterial reaction centre, focussing in particular on recent information on the structural consequences of site-directed mutagenesis, the roles played by water molecules in the reaction centre, the mechanism of ubiquinone reduction, and studies of the phospholipid environment of the protein.
Keywords: Reaction center; Photosynthesis; X-Ray crystallography; Mutagenesis; Water; Ubiquinone; Cardiolipin;
Succinate: quinone oxidoreductases: new insights from X-ray crystal structures by C.Roy D. Lancaster; A. Kröger (422-431).
Membrane-bound succinate dehydrogenases (succinate:quinone reductases, SQR) and fumarate reductases (quinol:fumarate reductases, QFR) couple the oxidation of succinate to fumarate to the reduction of quinone to quinol and also catalyse the reverse reaction. SQR (respiratory complex II) is involved in aerobic metabolism as part of the citric acid cycle and of the aerobic respiratory chain. QFR is involved in anaerobic respiration with fumarate as the terminal electron acceptor, and is part of an electron transport chain catalysing the oxidation of various donor substrates by fumarate. QFR and SQR complexes are collectively referred to as succinate:quinone oxidoreductases (EC 184.108.40.206), have very similar compositions and are predicted to share similar structures. The complexes consist of two hydrophilic and one or two hydrophobic, membrane-integrated subunits. The larger hydrophilic subunit A carries covalently bound flavin adenine dinucleotide and subunit B contains three iron-sulphur centres. QFR of Wolinella succinogenes and SQR of Bacillus subtilis contain only one hydrophobic subunit (C) with two haem b groups. In contrast, SQR and QFR of Escherichia coli contain two hydrophobic subunits (C and D) which bind either one (SQR) or no haem b group (QFR). The structure of W. succinogenes QFR has been determined at 2.2 Å resolution by X-ray crystallography (C.R.D. Lancaster, A. Kröger, M. Auer, H. Michel, Nature 402 (1999) 377–385). Based on this structure of the three protein subunits and the arrangement of the six prosthetic groups, a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b to the site of fumarate reduction and a mechanism of fumarate reduction was proposed. The W. succinogenes QFR structure is different from that of the haem-less QFR of E. coli, described at 3.3 Å resolution (T.M. Iverson, C. Luna-Chavez, G. Cecchini, D.C. Rees, Science 284 (1999) 1961–1966), mainly with respect to the structure of the membrane-embedded subunits and the relative orientations of soluble and membrane-embedded subunits. Also, similarities and differences between QFR transmembrane helix IV and transmembrane helix F of bacteriorhodopsin and their implications are discussed.
Keywords: Bioenergetics; Fumarate reductase; Membrane protein; Respiration; Succinate dehydrogenase; X-Ray crystallography;
The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity by James N Siedow; Ann L Umbach (432-439).
Mitochondria from all plants, many fungi and some protozoa contain a cyanide-resistant, alternative oxidase that functions in parallel with cytochrome c oxidase as the terminal oxidase on the electron transfer chain. Characterization of the structural and potential regulatory features of the alternative oxidase has advanced considerably in recent years. The active site is proposed to contain a di-iron center belonging to the ribonucleotide reductase R2 family and modeling of a four-helix bundle to accommodate this active site within the C-terminal two-thirds of the protein has been carried out. The structural features of this active site are conserved among all known alternative oxidases. The post-translational regulatory features of the alternative oxidase are more variable among organisms. The plant oxidase is dimeric and can be stimulated by either α-keto acids or succinate, depending upon the presence or absence, respectively, of a critical cysteine residue found in a conserved block of amino acids in the N-terminal region of the plant protein. The fungal and protozoan alternative oxidases generally exist as monomers and are not subject to organic acid stimulation but can be stimulated by purine nucleotides. The origins of these diverse regulatory features remain unknown but are correlated with sequence differences in the N-terminal third of the protein.
Keywords: Alternative oxidase; Mitochondrial electron transfer; Di-iron oxidase;
Crystallographic location of two Zn2+-binding sites in the avian cytochrome bc 1 complex by Edward A Berry; Zhaolei Zhang; Henry D Bellamy; Lishar Huang (440-448).
The chicken mitochondrial ubiquinol cytochrome c oxidoreductase (bc 1 complex) is inhibited by Zn2+ ions, but with higher K i (∼3 μM) than the corresponding bovine enzyme. When equilibrated with mother liquor containing 200 μM ZnCl2 for 7 days, the crystalline chicken bc 1 complex specifically binds Zn2+ at 4 sites representing two sites on each monomer in the dimer. These two sites are close to the stigmatellin-binding site, taken to be center Qo of the Q-cycle mechanism, and are candidates for the inhibitory site. One binding site is actually in the hydrophobic channel between the Qo site and the bulk lipid phase, and may interfere with quinone binding. The other is in a hydrophilic area between cytochromes b and c 1, and might interfere with the egress of protons from the Qo site to the intermembrane aqueous medium. No zinc was bound near the putative proteolytic active site of subunits 1 and 2 (homologous to mitochondrial processing peptidase) under these conditions.
Fusion protein approach to improve the crystal quality of cytochrome bo 3 ubiquinol oxidase from Escherichia coli by Bernadette Byrne; Jeff Abramson; Magnus Jansson; Erik Holmgren; So Iwata (449-455).
Crystals of cytochrome bo 3 ubiquinol oxidase from E. coli diffract X-rays to 3.5 Å and the structure determination is in progress. The limiting factor to the elucidation of the structural detail is the quality of the crystals; the diffraction spots from the crystals are diffused which leads to difficulties in processing the data beyond 4.0 Å. Weak protein–protein contacts within the crystal lattice is assumed to be the cause of this problem. To improve these contacts, we have introduced protein Z to the C-terminal end of the subunit IV of cytochrome bo 3 and expressed both proteins as a single fusion. We have successfully obtained crystals of this fusion protein. The spot shape problem has clearly been solved in the crystals of the fusion protein although further optimization is necessary to obtain higher resolution. We also discuss the potential applications of this approach to the crystallization of membrane proteins in general.
Keywords: Ubiquinol oxidase; Cytochrome bo 3; Crystallization; Membrane protein; Fusion protein;
Proton-coupled electron transfer at the Qo site: what type of mechanism can account for the high activation barrier? by Antony R. Crofts; Mariana Guergova-Kuras; Richard Kuras; Natalya Ugulava; Jiyuan Li; Sangjin Hong (456-466).
In Rhodobacter sphaeroides, transfer of the first electron in quinol oxidation by the bc 1 complex shows kinetic features (a slow rate (approx. 1.5×103/s), high activation energy (approx. 65 kJ/mol) and reorganization energy, λ (2.5 V)) that are unexpected from Marcus theory and the distances shown by the structures. Reduction of the oxidized iron-sulfur protein occurs after formation of the enzyme-substrate complex, and involves a H-transfer in which the electron transfer occurs through the approx. 7 Å of a bridging histidine forming a H-bond with quinol and a ligand to 2Fe-2S. The anomalous kinetic features can be explained by a mechanism in which the electron transfer is constrained by coupled transfer of the proton. We discuss this in the context of mutant strains with modified E m,7 and pK for the iron-sulfur protein, and Marcus theory for proton-coupled electron transfer. We suggest that transfer of the second proton and electron involve movement of semiquinone in the Qo site, and rotation of the Glu of the conserved -PEWY- sequence. Mutational studies show a key role for the domain proximal to heme b L. The effects of mutation at Tyr-302 (Tyr-279 in bovine sequence) point to a possible linkage between conformational changes in the proximal domain, and changes leading to closure of the iron-sulfur protein access channel at the distal domain.
Keywords: Myxothiazol; Stigmatellin; Activation energy; Mutagenesis; Proton-coupled electron transfer;
The cytochrome b 6 f complex: structural studies and comparison with the bc 1 complex by Cécile Breyton (467-474).
Electron crystallography of the chloroplastic b 6 f complex allowed the calculation of projection maps of crystals negatively stained or embedded in glucose. This gives insights into the overall structure of the extra- and transmembrane domains of the complex. A comparison with the structure of the bc 1 complex, the mitochondrial homologue of the b 6 f complex, suggests that the transmembrane domains of the two complexes are very similar, confirming the structural homology deduced from sequence analysis. On the other hand, the extramembrane organisation of the c-type cytochrome and of the Rieske protein seems quite different. Nevertheless, the same type of movement of the Rieske protein is observed in the b 6 f as in the bc 1 complex upon the binding of the quinol analogue stigmatellin. Crystallographic data also suggest movements in the transmembrane domains of the b 6 f complex, which would be specific of the b 6 f complex.
Keywords: Cytochrome b 6 f complex; Cytochrome bc 1 complex; Electron crystallography; Stigmatellin; Membrane protein; Chlamydomonas reinhardtii;
Protonation reactions in relation to the coupling mechanism of bovine cytochrome c oxidase by Peter R. Rich; Jacques Breton; Susanne Jünemann; Peter Heathcote (475-480).
Identification of the locations of protonatable sites in cytochrome c oxidase that are influenced by reactions in the binuclear centre is critical to assessment of proposed coupling mechanisms, and to controversies on where the pumping steps occur. One such protonation site is that which governs interconversion of the isoelectronic 607 nm ‘PM’ and 580 nm ‘F⋅’ forms of the two-electron-reduced oxygen intermediate. Low pH favours protonation of a site that is close to an electron paramagnetic resonance (EPR)-silent radical species in PM, and this induces a partial electronic redistribution to form an EPR-detectable tryptophan radical in F⋅. A further protonatable group that must be close to the binuclear centre has been detected in bacterial oxidases by Fourier transform infrared spectroscopy from pH-dependent changes in the haem-bound CO vibration frequency at low temperatures. However, in bovine cytochrome c oxidase under similar conditions of measurement, haem-bound CO remains predominantly in a single 1963 cm−1 form between pH 6.5 and 8.5, indicating that this group is not present. Lack of pH dependence extends to the protein region of the CO photolysis spectra and suggests that both the reduced and the reduced/CO states do not have titratable groups that affect the binuclear centre strongly in the pH range 6.5–8.5. This includes the conserved glutamic acid residue E242 whose pK appears to be above 8.5 even in the fully oxidised enzyme. The results are discussed in relation to recent ideas on coupling mechanism.
Keywords: Cytochrome c oxidase; Hydrogen peroxide; P state; F state; Electron paramagnetic resonance; Fourier transform infrared;
Towards understanding the chemistry of photosynthetic oxygen evolution: dynamic structural changes, redox states and substrate water binding of the Mn cluster in photosystem II by Johannes Messinger (481-488).
This mini-review summarizes my postdoctoral research in the labs of T. Wydrzynski/C.B. Osmond, J.H.A. Nugent/M.C.W. Evans and V.K. Yachandra/K. Sauer/M.P. Klein. The results are reported in the context of selected data from the literature. Special emphasis is given to the mode of substrate water binding, Mn oxidation states and the structures of the Mn cluster in the four (meta)stable redox states of the oxygen evolving complex. The paper concludes with a working model for the mechanism of photosynthetic water oxidation that combines μ-oxo bridge oxidation in the S3 state (V.K. Yachandra, K. Sauer, M.P. Klein, Chem. Rev. 96 (1996) 2927–2950) with O-O bond formation between two terminal Mn-hydroxo ligands during the S3→(S4)→S0 transition.
Keywords: Photosystem II; Water oxidation; Oxygen evolution; Manganese cluster;
Cloning and expression of cDNAs encoding plant V-ATPase subunits in the corresponding yeast null mutants by Keren Aviezer-Hagai; Hannah Nelson; Nathan Nelson (489-498).
Complementation of yeast null mutants is widely used for cloning of homologous genes from heterologous sources. We have used this method to clone the relevant V-ATPase genes from lemon fruit and Arabidopsis thaliana cDNA libraries. The pH levels are very different in the vacuoles of the lemon fruit and the A. thaliana, yet both are the result of the activity of the same enzyme complex, namely the V-ATPase. In order to investigate the mechanism that enables the enzyme to maintain such differences in pH values, we have compared the subunit composition of the V-ATPase complex from both sources. Towards this end, we have constructed a cDNA library from lemon fruit and cloned it into a similar shuttle vector to the one of the A. thaliana cDNA library, which is commercially available. In this work, we report the cloning and expression of VMA10 from both sources, two isoforms of the lemon proteolipid (VMA3) and the lemon homologue of yeast VPH1/STV1 subunit, LEMAC.
Keywords: V-ATPase; Lemon fruit; Arabidopsis thaliana; Expression cloning; cDNA library;
Biological nano motor, ATP synthase FoF1: from catalysis to γϵc 10–12 subunit assembly rotation by Yoh Wada; Yoshihiro Sambongi; Masamitsu Futai (499-505).
Proton translocating ATPase (ATP synthase), a chemiosmotic enzyme, synthesizes ATP from ADP and phosphate coupling with the electrochemical ion gradient across the membrane. This enzyme has been studied extensively by combined genetic, biochemical and biophysical approaches. Such studies revealed a unique mechanism which transforms an electrochemical ion gradient into chemical energy through the rotation of a subunit assembly. Thus, this enzyme can be defined as a nano motor capable of coupling a chemical reaction and ion translocation, or more simply, as a protein complex carrying out rotational catalysis. In this article, we briefly discuss our recent work, emphasizing the rotation of subunit assembly (γϵc 10–12) which is formed from peripheral and intrinsic membrane subunits.
Critical evaluation of the one- versus the two-channel model for the operation of the ATP synthase’s Fo motor by Peter Dimroth; Ulrich Matthey; Georg Kaim (506-513).
The mechanism of converting an electrochemical gradient of protons or Na+ ions across the membrane into rotational torque by the Fo motor of the ATP synthase has been described by a two-channel model or by a one-channel model. Experimental evidence obtained with the Fo motor from the Propionigenium modestum ATP synthase is described which is in accordance with the one-channel model, but not with the two-channel model. This evidence includes the ATP-dependent occlusion of one 22Na+ per ATP synthase with a mutated Na+-impermeable a subunit or the Na+ in/22Na+ out exchange which is not affected by modifying part of the c subunit sites with dicyclohexylcarbodiimide.
Keywords: ATP synthase; Rotational mechanism; Fo motor; Propionigenium modestum; Na+ occlusion; Na+ in/22Na+ out exchange;
The role of the D- and K-pathways of proton transfer in the function of the haem–copper oxidases by Mårten Wikström; Audrius Jasaitis; Camilla Backgren; Anne Puustinen; Michael I Verkhovsky (514-520).
The X-ray structures of several haem–copper oxidases now at hand have given important constraints on how these enzymes function. Yet, dynamic data are required to elucidate the mechanisms of electron and proton transfer, the activation of O2 and its reduction to water, as well as the still enigmatic mechanism by which these enzymes couple the redox reaction to proton translocation. Here, some recent observations will be briefly reviewed with special emphasis on the functioning of the so-called D- and K-pathways of proton transfer. It turns out that only one of the eight protons taken up by the enzyme during its catalytic cycle is transferred via the K-pathway. The D-pathway is probably responsible for the transfer of all other protons, including the four that are pumped across the membrane. The unique K-pathway proton may be specifically required to aid O–O bond scission by the haem–copper oxidases.
A re-examination of the structural and functional consequences of mutation of alanine-128 of the b subunit of Escherichia coli ATP synthase to aspartic acid by Stanley D Dunn; Yumin Bi; Matthew Revington (521-527).
The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F1 sector were analyzed. Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b 34–156. However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132. Unlike the wild-type sequence, the A128D mutant was unable to interact with F1-ATPase. These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F1 but having little or no effect on the dimeric nature of b.
Keywords: ATP Synthase; b subunit; Dimerization; Stalk; Analytical ultracentrifugation;
Light-induced FTIR difference spectroscopy of the S2-to-S3 state transition of the oxygen-evolving complex in Photosystem II by Hsiu-An Chu; Warwick Hillier; Neil A Law; Heather Sackett; Shannon Haymond; Gerald T Babcock (528-532).
We have applied flash-induced FTIR spectroscopy to study structural changes upon the S2-to-S3 state transition of the oxygen-evolving complex (OEC) in Photosystem II (PSII). We found that several modes in the difference IR spectrum are associated with bond rearrangements induced by the second laser flash. Most of these IR modes are absent in spectra of S2/S1, of the acceptor-side non-heme ion, of Y⋅ D/YD and of S3′/S2′ from Ca-depleted PSII preparations. Our results suggest that these IR modes most likely originate from structural changes in the oxygen-evolving complex itself upon the S2-to-S3 state transition in PSII.
Keywords: Fourier transform infrared spectroscopy; Photosystem II; Manganese cluster; Oxygen evolution;
Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase by Pia Ådelroth; Martin Karpefors; Gwen Gilderson; Farol L. Tomson; Robert B. Gennis; Peter Brzezinski (533-539).
We have investigated the electron–proton coupling during the peroxy (PR) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of CuA is increased by ∼100 mV, which slows electron transfer to the binuclear centre during the F→O transition by a factor of ∼200. In ED(I-286) proton uptake during PR→F was slowed by a factor of ∼5, which indicates that E(I-286) is the proton donor to PR. In addition, in the mutant enzyme the F→O transition rate displayed a deuterium isotope effect of ∼2.5 as compared with ∼7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Ådelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F→O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of ∼8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.
Keywords: Electron transfer; Proton transfer; Proton pumping; Cytochrome aa 3; Flash photolysis; Flow flash;