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BBA - Bioenergetics (v.1767, #9)
Infrared spectroscopy of proteins by Andreas Barth (pp. 1073-1101).
This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.
Keywords: Abbreviations; ATR; attenuated total reflection; δ; in plane bending vibration; FTIR; Fourier transform infrared; IR; infrared; γ; w; wagging vibration; γ; t; twisting vibration; γ; r; rocking vibration; ε; extinction coefficient; NMA; N; -methylacetamide; ν; stretching vibration; ν; s; symmetric stretching vibration; ν; as; antisymmetric stretching vibration; TDC; transition dipole coupling; TDM; transition dipole momentInfrared spectroscopy; FTIR; Protein; Protein structure; Protein function; Secondary structure; Water; Amide I; Amino acid side chain; Enzyme activity; Difference spectroscopy
Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase by Anne Tuukkanen; Ville R.I. Kaila; Liisa Laakkonen; Gerhard Hummer; Mårten Wikström (pp. 1102-1106).
In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a 3/CuB site, and for proton pumping. When present, the side chain of Glu-242 is orientated “down” towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity “above” Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an “up” position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the “up” position is preferred energetically when the cavity contains four water molecules, but the “down” position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the “resting” state of the enzyme, and that the “up/down” equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O2 reduction.
Keywords: Molecular dynamics; Nonpolar cavity; Proton transfer; Proton pump; Water
Interaction of heme and heme–hemopexin with an extracellular oxidant system used to measure cell growth-associated plasma membrane electron transport by Kimberly R. Rish; Ryan Swartzlander; Takrima N. Sadikot; Michael V. Berridge; Ann Smith (pp. 1107-1117).
Since redox active metals are often transported across membranes into cells in the reduced state, we have investigated whether exogenous ferri-heme or heme bound to hemopexin (HPX), which delivers heme to cells via receptor-mediated endocytosis, interact with a cell growth-associated plasma membrane electron transport (PMET) pathway. PMET reduces the cell-impermeable tetrazolium salt, WST-1, in the presence of the mandatory low potential intermediate electron acceptor, mPMS. In human promyelocytic (HL60) cells, protoheme (iron protoporphyrin IX; 2,4-vinyl), mesoheme (2,4-ethyl) and deuteroheme (2,4-H) inhibited reduction of WST-1/ mPMS in a saturable manner supporting interaction with a finite number of high affinity acceptor sites (Kd 221 nM for naturally occurring protoheme). A requirement for the redox-active iron was shown using gallium-protoporphyrin IX (PPIX) and tin-PPIX. Heme–hemopexin, but not apo-hemopexin, also inhibited WST-1 reduction, and copper was required. Importantly, since neither heme nor heme–hemopexin replace mPMS as an intermediate electron acceptor and since inhibition of WST-1/mPMS reduction requires living cells, the experimental evidence supports the view that heme and heme–hemopexin interact with electrons from PMET. We therefore propose that heme and heme–hemopexin are natural substrates for this growth-associated electron transfer across the plasma membrane.
Keywords: PMET; Heme; Hemopexin; Plasma membrane electron transport; Oxidoreductase
Dopamine enhances mtNOS activity: Implications in mitochondrial function by Analía Czerniczyniec; Juanita Bustamante; Silvia Lores-Arnaiz (pp. 1118-1125).
Dopamine and nitric oxide systems can interact in different processes in the central nervous system. Dopamine and oxidation products have been related to mitochondrial dysfunction. In the present study, intact mitochondria and submitochondrial membranes were incubated with different DA concentrations for 5 min. Dopamine (1 mM) increased nitric oxide production in submitochondrial membranes and this effect was partially prevented in the presence of both DA and NOS inhibitor Nω-nitro-l-arginine (l-NNA). A 46% decrease in state 3 oxygen uptake (active respiration state) was found after 15 mM dopamine incubation. When mitochondria were incubated with 15 mM dopamine in the presence ofl-NNA, state 3 respiratory rate was decreased by only 17% showing the involvement of NO. As shown for O2 consumption, the inhibition of cytochrome oxidase by 1 mM DA was mediated by NO. Hydrogen peroxide production significantly increased after 15 mM DA incubation, being mainly due to its metabolism by MAO. Also, DA-induced depolarization was prevented by the addition ofl-NNA showing the involvement of nitric oxide in this process too. This work provides evidence that in the studied conditions, dopamine modifies mitochondrial function by a nitric oxide-dependent pathway.
Keywords: Dopamine; Mitochondrial respiration; Brain mitochondria; Nitric oxide; Mitochondrial membrane potential
Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation by Paula B.M. Luís; Jos P.N. Ruiter; Cátia C.P. Aires; Graça Soveral; Isabel Tavares de Almeida; Marinus Duran; Ronald J.A. Wanders; Margarida F.B. Silva (pp. 1126-1133).
The effect of the antiepileptic drug valproic acid (VPA) on mitochondrial oxidative phosphorylation (OXPHOS) was investigated in vitro. Two experimental approaches were used, in the presence of selected respiratory-chain substrates: (1) formation of ATP in digitonin permeabilized rat hepatocytes and (2) measurement of the rate of oxygen consumption by polarography in rat liver mitochondria. VPA (0.1–1.0 mM) was found to inhibit oxygen consumption and ATP synthesis under state 3 conditions with glutamate and 2-oxoglutarate as respiratory substrates. No inhibitory effect on OXPHOS was observed when succinate (plus rotenone) was used as substrate. We tested the hypothesis that dihydrolipoyl dehydrogenase (DLDH) might be a direct target of VPA, especially its acyl-CoA intermediates. Valproyl-CoA (0.5–1.0 mM) and valproyl-dephosphoCoA (0.5–1.0 mM) both inhibited the DLDH activity, acting apparently by different mechanisms. The decreased activity of DLDH induced by VPA metabolites may, at least in part, account for the impaired rate of oxygen consumption and ATP synthesis in mitochondria if 2-oxoglutarate or glutamate were used as respiratory substrates, thus limiting the flux of these substrates through the citric acid cycle.
Keywords: Abbreviations; VPA; 2-; n; -propylpentanoic acid or valproic acid; Δ; 2(E); -VPA; 2-; n; -propyl-2-pentenoic acid; Δ; 4; -VPA; 2-; n; -propyl-4-pentenoic acid; CoA; coenzyme A; dephCoA; dephosphoCoA; EGTA; ethylene glycol-bis(β-aminoethyl ether)-; N,N,N′,N′; ,-tetraacetic acid; SEM buffer; Sucrose/EGTA/MOPS buffer; ADP; adenosine-5′-diphosphate; ATP; adenosine-5′-triphosphate; BCA; bicinchoninic acid; PDHC; pyruvate dehydrogenase complex; 2-OGDHC; 2-oxoglutarate dehydrogenase complex; BCODHC; branched chain 2-oxoacid dehydrogenase complex; DLDH; dihydrolipoyl dehydrogenase (dihydrolipoamide dehydrogenase or subunit E; 3; : EC 1.8.1.4)Valproic acid; Oxidative phosphorylation; Dihydrolipoyl dehydrogenase; Valproyl-CoA; Mitochondrial dysfunction; Drug-induced hepatotoxicity
PPARα-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation by Naomi van Vlies; Sacha Ferdinandusse; Marjolein Turkenburg; Ronald J.A. Wanders; Frédéric M. Vaz (pp. 1134-1142).
In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPARα−/− mice, indicating that PPARα is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPARα-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPARα−/− mice. Here we show that both enhancement of carnitine biosynthesis (due to increased γ-butyrobetaine dioxygenase activity), extra-hepatic γ-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPARα-dependent.
Keywords: Abbreviations; PPARα; Peroxisome proliferator activated receptor α; TML; 6-N-trimethyllysine; TMLD; 6-N-trimethyllysine dioxygenase; HTML; 3-hydroxy-6-N-trimethyllysine; TMABA; 4-trimethylaminobutyraldehyde; TMABA-DH; 4-trimethylaminobutyraldehyde dehydrogenase; γ-BB; 4-trimethylaminobutyric acid; γ-BBD; 4-trimethylaminobutyric acid dioxygenaseCarnitine; PPARα; Carnitine biosynthesis; OCTN2; γ-butyrobetaine dioxygenase; fasting
Energy diagrams and mechanism for proton pumping in cytochrome c oxidase by Per E.M. Siegbahn; Margareta R.A. Blomberg (pp. 1143-1156).
The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a3 as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and p Ka values during the pumping process are derived for comparison to experiments.
Keywords: Cytochrome c oxidase; Theory; Proton pumping; Diagram; Mechanism
Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease by Adrienne DeCorby; Dana Gášková; Leanne C. Sayles; Bernard D. Lemire (pp. 1157-1163).
The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.
Keywords: Mitochondria; Membrane potential; Nematode; NADH:ubiquinone oxidoreductase; DiS-C; 3; (3)
Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria by Inna I. Severina; Mikhail Yu. Vyssokikh; Antonina V. Pustovidko; Ruben A. Simonyan; Tatiana I. Rokitskaya; Vladimir P. Skulachev (pp. 1164-1168).
In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199–208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (ΔΨ), the compartment with a lower dication concentration positive. However, the ΔΨ values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at ΔΨ >100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian ΔΨ, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered ΔΨ. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.
Keywords: Dication; Lipophilic cation; Planar phospholipids membrane; Mitochondria; Membrane potential; Damaging effectAbbreviations; ΔΨ; transmembrane electric potential difference; BLM; bilayer planar phospholipid membrane; BSA; bovine serum albumin; DNP; 2,4-; p; -dinitrophenol; P2C5 and P2C10; bistriphenylphosphonium with five or ten methylene linker, respectively; TPMP; N,N; ′-tetramethyl-; p; -phenylenediamine; TPB; tetraphenyl borate; TPP; tetraphenylphosphonium
Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3 by Catarina M. Paquete; David L. Turner; Ricardo O. Louro; António V. Xavier; Teresa Catarino (pp. 1169-1179).
The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c3 isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem–haem and haem–proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.
Keywords: Abbreviations; TpI; c; 3; Type I cytochrome; c; 3; TpII; c; 3; Type II cytochrome; c; 3; D.; Desulfovibrio; Dsm.; Desulfomicrobium; NMR; Nuclear Magnetic ResonanceCytochrome c; 3; Thermodynamic characterisation; Electron transfer kinetics; Multicentre protein; Desulfovibrio
Structural and functional self-organization of Photosystem II in grana thylakoids by Helmut Kirchhoff; Winfried Haase; Silvia Haferkamp; Thomas Schott; Mauricio Borinski; Ulrich Kubitscheck; Matthias Rögner (pp. 1180-1188).
The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.
Keywords: Abbreviations; Chl; chlorophyll; DCMU; 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; cyt; cytochrom; EDTA; ethylenediaminetetraacetic acid; EF; exoplasmatic fracture face; Fo; chlorophyll a fluorescence level with oxidized QA; Fm; fluorescence level with completely reduced QA; HEPES; N-2-hydroxyethylpiperazine-; N′; -2-ethane-sulfonic acid; LHC; light harvesting complex; NNDF; next neighbor distribution function; PCF; pair correlation function; PF; protoplasmatic fracture face; PS; photosystem; QA; primary quinone acceptor of photosystem II; RC; reaction center; rs; restacked; s; stacked; us; unstackedGrana thylakoid; PSII; Repair cycle; Stacking