BBA - Bioenergetics (v.1837, #10)

A simple method to engineer a protein-derived redox cofactor for catalysis by Sooim Shin; Moonsung Choi; Heather R. Williamson; Victor L. Davidson (1595-1601).
The 6 ×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co2 +. Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co2 +-loaded 6 × His-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co2 +-loaded 6 × His-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co2 +-loaded 6 × His-tagged Cu1 + amicyanin oxidizes the copper site which is 20 Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.Display Omitted
Keywords: Protein Engineering; Enzyme; Biotechnology; Bioenergetics; Histidine tag;

Modulation of the mitochondrial large-conductance calcium-regulated potassium channel by polyunsaturated fatty acids by Anna Olszewska; Piotr Bednarczyk; Detlef Siemen; Adam Szewczyk (1602-1610).
Polyunsaturated fatty acids (PUFAs) and their metabolites can modulate several biochemical processes in the cell and thus prevent various diseases. PUFAs have a number of cellular targets, including membrane proteins. They can interact with plasma membrane and intracellular potassium channels. The goal of this work was to verify the interaction between PUFAs and the most common and intensively studied mitochondrial large conductance Ca2+-regulated potassium channel (mitoBKCa). For this purpose human astrocytoma U87 MG cell lines were investigated using a patch-clamp technique. We analyzed the effects of arachidonic acid (AA); eicosatetraynoic acid (ETYA), which is a non-metabolizable analog of AA; docosahexaenoic acid (DHA); and eicosapentaenoic acid (EPA). The open probability (Po) of the channel did not change significantly after application of 10 μM ETYA. Po increased, however, after adding 10 μM AA. The application of 30 μM DHA or 10 μM EPA also increased the Po of the channel. Additionally, the number of open channels in the patch increased in the presence of 30 μM EPA. Collectively, our results indicate that PUFAs regulate the BKCa channel from the inner mitochondrial membrane.
Keywords: Mitochondria; Fatty acids; Large-conductance calcium-activated potassium channel;

Engineered biosynthesis of bacteriochlorophyll b in Rhodobacter sphaeroides by Daniel P. Canniffe; C. Neil Hunter (1611-1616).
Bacteriochlorophyll b has the most red-shifted absorbance maximum of all naturally occurring photopigments. It has a characteristic ethylidene group at the C8 position in place of the more common ethyl group, the product of a C8-vinyl reductase, which is carried by the majority of chlorophylls and bacteriochlorophylls used in photosynthesis. The subsequent and first step exclusive to bacteriochlorophyll biosynthesis, the reduction of the C7 = C8 bond, is catalyzed by chlorophyllide oxidoreductase. It has been demonstrated that the enzyme from bacteriochlorophyll a-utilizing bacteria can catalyze the formation of compounds carrying an ethyl group at C8 from both ethyl- and vinyl-carrying substrates, indicating a surprising additional C8-vinyl reductase function, while the enzyme from organisms producing BChl b could only catalyze C7 = C8 reduction with a vinyl substrate, but this product carried an ethylidene group at the C8 position. We have replaced the native chlorophyllide oxidoreductase-encoding genes of Rhodobacter sphaeroides with those from Blastochloris viridis, but the switch from bacteriochlorophyll a to b biosynthesis is only detected when the native conventional C8-vinyl reductase is absent. We propose a non-enzymatic mechanism for ethylidene group formation based on the absence of cellular C8-vinyl reductase activity.
Keywords: Bacteriochlorophyll; Chlorophyll; Chlorophyllide oxidoreductase; Photosynthesis; Pathway engineering;

Ca2 + regulation of mitochondrial function in neurons by Carlos B. Rueda; Irene Llorente-Folch; Ignacio Amigo; Laura Contreras; Paloma González-Sánchez; Paula Martínez-Valero; Inés Juaristi; Beatriz Pardo; Araceli del Arco; Jorgina Satrústegui (1617-1624).
Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca2 + signal or to Ca2 + itself. [Na+]i, [Ca2 +]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca2 + clearly showed that Ca2 +-stimulation of coupled respiration is required to maintain [ATP]i levels. Ca2 + may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca2 + entry in mitochondria through the calcium uniporter (MCU). Two Ca2 +-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate–glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate–aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca2 + of 300 nM and 3.4 μM, respectively. The lack of SCaMC-3 results in a smaller Ca2 +-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca2 +-dependent responses to all workloads: a reduction of about 65–70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K+-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K+-depolarization, this occurs in spite of the presence of large [Ca2 +]mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca2 +-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca2 + is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.Display Omitted
Keywords: Calcium; Mitochondrion; Aspartate–glutamate transporter; ATP-Mg/Pi transporter; Neuronal respiration; Calcium-regulated transport;

The functional or regulatory role of long-distance interactions between protein surface and interior represents an insufficiently understood aspect of protein function. Cationic screening of surface charges determines the morphology of thylakoid membrane stacks. We show that it also influences directly the light-driven reactions in the interior of photosystem II (PSII). After laser-flash excitation of PSII membrane particles from spinach, time courses of the delayed recombination fluorescence (10 μs–10 ms) and the variable chlorophyll-fluorescence yield (100 μs–1 s) were recorded in the presence of chloride salts. At low salt-concentrations, a stimulating effect was observed for the S-state transition efficiency, the time constant of O2-formation at the Mn4Ca-complex of PSII, and the halftime of re-oxidation of the primary quinone acceptor (Qa) by the secondary quinone acceptor (Qb). The cation valence determined the half-effect concentrations of the stimulating salt effect, which were around 6 μM, 200 μM and 10 mM for trivalent (LaCl3), bivalent (MgCl2, CaCl2), and monovalent cations (NaCl, KCl), respectively. A depressing high-salt effect also depended strongly on the cation valence (onset concentrations around 2 mM, 50 mM, and 500 mM). These salt effects are proposed to originate from electrostatic screening of negatively charged carboxylate sidechains, which are found in the form of carboxylate clusters at the solvent-exposed protein surface. We conclude that the influence of electrostatic screening by solvent cations manifests a functionally relevant long-distance interaction between protein surface and electron-transfer reactions in the protein interior. A relation to regulation and adaptation in response to environmental changes is conceivable.
Keywords: Chlorophyll fluorescence; Electrostatic screening; Manganese complex; Oxygen evolution; Photosynthesis; Water oxidation;

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography by Misha Kudryashev; Aikaterini Aktoudianaki; Dimitrios Dedoglou; Henning Stahlberg; Georgios Tsiotis (1635-1642).
Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl α-containing Fenna–Matthews–Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane.
Keywords: Electron microscopy; Cryo-electron tomography; Green sulfur bacteria; Chlorosome;

Superoxide production by cytochrome bc 1 complex: A mathematical model by F. Guillaud; S. Dröse; A. Kowald; U. Brandt; E. Klipp (1643-1652).
Reactive oxygen species (ROS) are involved in the pathophysiology of several diseases (e.g. Alzheimer or atherosclerosis) and also in the aging process. The main source of ROS in aerobic organisms is the electron transport chain (ETC) in the inner mitochondrial membrane. Superoxide is produced at complexes I and III of the ETC, starting a complex network of ROS reactions. To achieve a deeper mechanistic understanding of how ROS are generated by complex III, we developed a mathematical model that successfully describes experimental data of complex III activity in various rat tissues, the production of ROS with and without antimycin and ROS generation depending on different values of the membrane potential ∆Ψ. The model also reinforces the idea of ubiquinone acting as a redox mediator between heme b L and oxygen, as proposed earlier.
Keywords: Mathematical model; Complex III; Reactive oxygen species; Superoxide; Antimycin A;

A phycocyanin-deletion mutant of Synechocystis (cyanobacteria) was generated upon replacement of the CPC-operon with a kanamycin resistance cassette. The Δcpc transformant strains (Δcpc) exhibited a green phenotype, compared to the blue-green of the wild type (WT), lacked the distinct phycocyanin absorbance at 625 nm, and had a lower Chl per cell content and a lower PSI/PSII reaction center ratio compared to the WT. Molecular and genetic analyses showed replacement of all WT copies of the Synechocystis DNA with the transgenic version, thereby achieving genomic DNA homoplasmy. Biochemical analyses showed the absence of the phycocyanin α- and β-subunits, and the overexpression of the kanamycin resistance NPTI protein in the Δcpc. Physiological analyses revealed a higher, by a factor of about 2, intensity for the saturation of photosynthesis in the Δcpc compared to the WT. Under limiting intensities of illumination, growth of the Δcpc was slower than that of the WT. This difference in the rate of cell duplication diminished gradually as growth irradiance increased. Identical rates of cell duplication of about 13 h for both WT and Δcpc were observed at about 800 μmol photons m− 2  s− 1 or greater. Culture productivity analyses under simulated bright sunlight and high cell-density conditions showed that biomass accumulation by the Δcpc was 1.57-times greater than that achieved by the WT. Thus, the work provides first-time direct evidence of the applicability of the Truncated Light-harvesting Antenna (TLA)-concept in cyanobacteria, entailing substantial improvements in the photosynthetic efficiency and productivity of mass cultures upon minimizing the phycobilisome light-harvesting antenna size.
Keywords: Cyanobacterium; Phycocyanin deletion; Photosynthesis; Phycobilisome; Productivity; TLA concept;

Organization in photosynthetic membranes of purple bacteria in vivo: The role of carotenoids by Sandrine E. D'Haene; Lucy I. Crouch; Michael R. Jones; Raoul N. Frese (1665-1673).
Photosynthesis in purple bacteria is performed by pigment–protein complexes that are closely packed within specialized intracytoplasmic membranes. Here we report on the influence of carotenoid composition on the organization of RC–LH1 pigment–protein complexes in intact membranes and cells of Rhodobacter sphaeroides. Mostly dimeric RC–LH1 complexes could be isolated from strains expressing native brown carotenoids when grown under illuminated/anaerobic conditions, or from strains expressing green carotenoids when grown under either illuminated/anaerobic or dark/semiaerobic conditions. However, mostly monomeric RC–LH1 complexes were isolated from strains expressing the native photoprotective red carotenoid spheroidenone, which is synthesized during phototrophic growth in the presence of oxygen. Despite this marked difference, linear dichroism (LD) and light-minus-dark LD spectra of oriented intact intracytoplasmic membranes indicated that RC–LH1 complexes are always assembled in ordered arrays, irrespective of variations in the relative amounts of isolated dimeric and monomeric RC–LH1 complexes. We propose that part of the photoprotective response to the presence of oxygen mediated by synthesis of spheroidenone may be a switch of the structure of the RC–LH1 complex from dimers to monomers, but that these monomers are still organized into the photosynthetic membrane in ordered arrays. When levels of the dimeric RC–LH1 complex were very high, and in the absence of LH2, LD and ∆LD spectra from intact cells indicated an ordered arrangement of RC–LH1 complexes. Such a degree of ordering implies the presence of highly elongated, tubular membranes with dimensions requiring orientation along the length of the cell and in a proportion larger than previously observed.
Keywords: Photosynthetic membranes; Supramolecular organization; Linear dichroism spectroscopy; Carotenoid; Photoprotection; Rhodobacter sphaeroides;

Mitochondrial EF4 links respiratory dysfunction and cytoplasmic translation in Caenorhabditis elegans by Fang Yang; Yanyan Gao; Zhikai Li; Luming Chen; Zhiping Xia; Tao Xu; Yan Qin (1674-1683).
How animals coordinate cellular bioenergetics in response to stress conditions is an essential question related to aging, obesity and cancer. Elongation factor 4 (EF4/LEPA) is a highly conserved protein that promotes protein synthesis under stress conditions, whereas its function in metazoans remains unknown. Here, we show that, in Caenorhabditis elegans, the mitochondria-localized CeEF4 (referred to as mtEF4) affects mitochondrial functions, especially at low temperature (15 °C). At worms' optimum growing temperature (20 °C), mtef4 deletion leads to self-brood size reduction, growth delay and mitochondrial dysfunction. Transcriptomic analyses show that mtef4 deletion induces retrograde pathways, including mitochondrial biogenesis and cytoplasmic translation reorganization. At low temperature (15 °C), mtef4 deletion reduces mitochondrial translation and disrupts the assembly of respiratory chain supercomplexes containing complex IV. These observations are indicative of the important roles of mtEF4 in mitochondrial functions and adaptation to stressful conditions.Display Omitted
Keywords: EF4(LepA/GUF1); C. elegans; Mitochondrial dysfunction; Retrograde pathways; Translation;

In vitro analysis of the plastid terminal oxidase in photosynthetic electron transport by Kathleen Feilke; Qiuju Yu; Peter Beyer; Pierre Sétif; Anja Krieger-Liszkay (1684-1690).
The plastid terminal oxidase PTOX catalyzes the oxidation of plastoquinol (PQH2) coupled with the reduction of oxygen to water. In vivo PTOX is attached to the thylakoid membrane. PTOX is important for plastid development and carotenoid biosynthesis, and its role in photosynthesis is controversially discussed. To analyze PTOX activity in photosynthetic electron transport recombinant purified PTOX fused to the maltose-binding protein was added to photosystem II-enriched membrane fragments. These membrane fragments contain the plastoquinone (PQ) pool as verified by thermoluminescence. Experimental evidence for PTOX oxidizing PQH2 is demonstrated by following chlorophyll fluorescence induction. Addition of PTOX to photosystem II-enriched membrane fragments led to a slower rise, a lower level of the maximal fluorescence and an acceleration of the fluorescence decay. This effect was only observed at low light intensities indicating that PTOX cannot compete efficiently with the reduction of the PQ pool by photosystem II at higher light intensities. PTOX attached tightly to the membranes since it was only partly removable by membrane washings. Divalent cations enhanced the effect of PTOX on chlorophyll fluorescence compared to NaCl most likely because they increase connectivity between photosystem II centers and the size of the PQ pool. Using single turnover flashes, it was shown that the level of reactive oxygen species, generated by PTOX in a side reaction, increased when the spacing between subsequent double flashes was enlarged. This shows that PTOX generates reactive oxygen species under limited substrate availability.
Keywords: Plastid terminal oxidase; Chlorophyll fluorescence; Photosynthetic electron transport; Reactive oxygen species;

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS by Roland Tengölics; Lívia Mészáros; E. Győri; Zsolt Doffkay; Kornél L. Kovács; Gábor Rákhely (1691-1698).
Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.
Keywords: Hydrogenases; Electron transport; Sulfur metabolism; Thiocapsa roseopersicina; Photosynthesis; Photosynthetic bacteria;

Imaging mass spectrometry reveals fiber-specific distribution of acetylcarnitine and contraction-induced carnitine dynamics in rat skeletal muscles by Yasuro Furuichi; Naoko Goto-Inoue; Yasuko Manabe; Mitsutoshi Setou; Kazumi Masuda; Nobuharu L. Fujii (1699-1706).
Carnitine is well recognized as a key regulator of long-chain fatty acyl group translocation into the mitochondria. In addition, carnitine, as acetylcarnitine, acts as an acceptor of excess acetyl-CoA, a potent inhibitor of pyruvate dehydrogenase. Here, we provide a new methodology for accurate quantification of acetylcarnitine content and determination of its localization in skeletal muscles. We used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI–IMS) to visualize acetylcarnitine distribution in rat skeletal muscles. MALDI–IMS and immunohistochemistry of serial cross-sections showed that acetylcarnitine was enriched in the slow-type muscle fibers. The concentration of ATP was lower in muscle regions with abundant acetylcarnitine, suggesting a relationship between acetylcarnitine and metabolic activity. Using our novel method, we detected an increase in acetylcarnitine content after muscle contraction. Importantly, this increase was not detected using traditional biochemical assays of homogenized muscles. We also demonstrated that acetylation of carnitine during muscle contraction was concomitant with glycogen depletion. Our methodology would be useful for the quantification of acetylcarnitine and its contraction-induced kinetics in skeletal muscles.
Keywords: Carnitine; Muscle contraction; Fiber type;

Modeling the respiratory chain complexes with biothermokinetic equations — The case of complex I by Margit Heiske; Christine Nazaret; Jean-Pierre Mazat (1707-1716).
The mitochondrial respiratory chain plays a crucial role in energy metabolism and its dysfunction is implicated in a wide range of human diseases. In order to understand the global expression of local mutations in the rate of oxygen consumption or in the production of adenosine triphosphate (ATP) it is useful to have a mathematical model in which the changes in a given respiratory complex are properly modeled. Our aim in this paper is to provide thermodynamics respecting and structurally simple equations to represent the kinetics of each isolated complexes which can, assembled in a dynamical system, also simulate the behavior of the respiratory chain, as a whole, under a large set of different physiological and pathological conditions. On the example of the reduced nicotinamide adenine dinucleotide (NADH)–ubiquinol–oxidoreductase (complex I) we analyze the suitability of different types of rate equations. Based on our kinetic experiments we show that very simple rate laws, as those often used in many respiratory chain models, fail to describe the kinetic behavior when applied to a wide concentration range. This led us to adapt rate equations containing the essential parameters of enzyme kinetic, maximal velocities and Henri–Michaelis–Menten like-constants (KM and KI) to satisfactorily simulate these data.Display Omitted
Keywords: Complex I; Oxidative phosphorylation; Modeling;

Cytochrome c 1 exhibits two binding sites for cytochrome c in plants by Blas Moreno-Beltrán; Antonio Díaz-Quintana; Katiuska González-Arzola; Adrián Velázquez-Campoy; Miguel A. De la Rosa; Irene Díaz-Moreno (1717-1729).
In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c 1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c 1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome.
Keywords: Arabidopsis thaliana; Cytochrome c; Cytochrome bc 1 complex; NMR; Respirasome; Supercomplex;

Dynamics of the active site architecture in plant-type ferredoxin-NADP+ reductases catalytic complexes by Ana Sánchez-Azqueta; Daniela L. Catalano-Dupuy; Arleth López-Rivero; María Laura Tondo; Elena G. Orellano; Eduardo A. Ceccarelli; Milagros Medina (1730-1738).
Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP+ reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP+ coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor–acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.
Keywords: Ferredoxin-NADP+ reductase; Flavoenzyme; Kinetic isotope effect; Hydride transfer; Charge–transfer complex; Plastidic-type FNR; Bacterial-type FPR;

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector by Ljudmila S. Khailova; Denis N. Silachev; Tatyana I. Rokitskaya; Armine V. Avetisyan; Konstantin G. Lyamsaev; Inna I. Severina; Tatyana M. Il'yasova; Mikhail V. Gulyaev; Vera I. Dedukhova; Tatyana A. Trendeleva; Egor Y. Plotnikov; Renata A. Zvyagilskaya; Boris V. Chernyak; Dmitry B. Zorov; Yuri N. Antonenko; Vladimir P. Skulachev (1739-1747).
Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H+ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.
Keywords: Mitochondria; Mild uncoupler; Protonophore; Rhodamine; Membrane potential;

Highly efficient energy transfer from a carbonyl carotenoid to chlorophyll a in the main light harvesting complex of Chromera velia by Milan Durchan; Gürkan Keşan; Václav Šlouf; Marcel Fuciman; Hristina Staleva; Josef Tichý; Radek Litvín; David Bína; František Vácha; Tomáš Polívka (1748-1755).
We report on energy transfer pathways in the main light-harvesting complex of photosynthetic relative of apicomplexan parasites, Chromera velia. This complex, denoted CLH, belongs to the family of FCP proteins and contains chlorophyll (Chl) a, violaxanthin, and the so far unidentified carbonyl carotenoid related to isofucoxanthin. The overall carotenoid-to-Chl-a energy transfer exhibits efficiency over 90% which is the largest among the FCP-like proteins studied so far. Three spectroscopically different isofucoxanthin-like molecules were identified in CLH, each having slightly different energy transfer efficiency that increases from isofucoxanthin-like molecules absorbing in the blue part of the spectrum to those absorbing in the reddest part of spectrum. Part of the energy transfer from carotenoids proceeds via the ultrafast S2 channel of both the violaxanthin and isofucoxanthin-like carotenoid, but major energy transfer pathway proceeds via the S1/ICT state of the isofucoxanthin-like carotenoid. Two S1/ICT-mediated channels characterized by time constants of ~ 0.5 and ~ 4 ps were found. For the isofucoxanthin-like carotenoid excited at 480 nm the slower channel dominates, while those excited at 540 nm employs predominantly the fast 0.5 ps channel. Comparing these data with the excited-state properties of the isofucoxanthin-like carotenoid in solution we conclude that, contrary to other members of the FCP family employing carbonyl carotenoids, CLH complex suppresses the charge transfer character of the S1/ICT state of the isofucoxanthin-like carotenoid to achieve the high carotenoid-to-Chl-a energy transfer efficiency.
Keywords: Light-harvesting; Energy transfer; Carbonyl carotenoids; Apicomplexa; FCP;

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells by Wojciech Giera; Sebastian Szewczyk; Michael D. McConnell; Joris Snellenburg; Kevin E. Redding; Rienk van Grondelle; Krzysztof Gibasiewicz (1756-1768).
Identical time-resolved fluorescence measurements with ~ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI–LHCI complexes and PSI–LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6–8 ps, 25–30 ps, 166–314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI–LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12–15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI–LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.
Keywords: Photosystem I; Light harvesting complex I; Chlamydomonas reinhardtii; Time-resolved fluorescence; Streak camera; Red chlorophylls;

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides by Michaël L. Cartron; John D. Olsen; Melih Sener; Philip J. Jackson; Amanda A. Brindley; Pu Qian; Mark J. Dickman; Graham J. Leggett; Klaus Schulten; C. Neil Hunter (1769-1780).
Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc 1 (cytbc 1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc 1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc 1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc 1 complexes occur as dimers in the membrane. The cytbc 1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc 1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc 1 whereas LH2 complexes became detached, consistent with clusters of cytbc 1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc 1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc 1, ATP synthase, cytaa 3 and cytcbb 3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc 1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.
Keywords: Bacterial photosynthesis; Cytochrome bc 1; Atomic force microscopy; Electron microscopy; Quinone; Membrane modelling;

Cytotoxicity of mitochondria-targeted resveratrol derivatives: Interactions with respiratory chain complexes and ATP synthase by Nicola Sassi; Andrea Mattarei; Michele Azzolini; Ildiko' Szabo'; Cristina Paradisi; Mario Zoratti; Lucia Biasutto (1781-1789).
We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low μM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H2O2 produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. “Capping” free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca2 +] “spikes” in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F0F1 ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore.
Keywords: Resveratrol; Triphenylphosphonium; ROS; Mitochondria; Respiratory chain; ATP synthase;

Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury by C. Quoilin; A. Mouithys-Mickalad; S. Lécart; M.-P. Fontaine-Aupart; M. Hoebeke (1790-1800).
To investigate the role of oxidative stress and/or mitochondrial impairment in the occurrence of acute kidney injury (AKI) during sepsis, we developed a sepsis-induced in vitro model using proximal tubular epithelial cells exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between oxidative stress and mitochondrial respiratory chain activity defects.LPS treatment resulted in an increase in the expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 4 (NOX-4), suggesting the cytosolic overexpression of nitric oxide and superoxide anion, the primary reactive nitrogen species (RNS) and reactive oxygen species (ROS). This oxidant state seemed to interrupt mitochondrial oxidative phosphorylation by reducing cytochrome c oxidase activity. As a consequence, disruptions in the electron transport and the proton pumping across the mitochondrial inner membrane occurred, leading to a decrease of the mitochondrial membrane potential, a release of apoptotic-inducing factors and a depletion of adenosine triphosphate. Interestingly, after being targeted by RNS and ROS, mitochondria became in turn producer of ROS, thus contributing to increase the mitochondrial dysfunction.The role of oxidants in mitochondrial dysfunction was further confirmed by the use of iNOS inhibitors or antioxidants that preserve cytochrome c oxidase activity and prevent mitochondrial membrane potential dissipation. These results suggest that sepsis-induced AKI should not only be regarded as failure of energy status but also as an integrated response, including transcriptional events, ROS signaling, mitochondrial activity and metabolic orientation such as apoptosis.
Keywords: Sepsis; Acute kidney injury; Mitochondrial dysfunction; Cytopathic hypoxia; Oxidative stress; Nitric oxide;

Kinetics of substrate inhibition of periplasmic nitrate reductase by Julien G.J. Jacques; Bénédicte Burlat; Pascal Arnoux; Monique Sabaty; Bruno Guigliarelli; Christophe Léger; David Pignol; Vincent Fourmond (1801-1809).
Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.Display Omitted
Keywords: Enzyme kinetics; Nitrate reductase; Electrochemistry; EPR spectroscopy;

Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria by Minenosuke Matsutani; Kota Fukushima; Chiho Kayama; Misato Arimitsu; Hideki Hirakawa; Hirohide Toyama; Osao Adachi; Toshiharu Yakushi; Kazunobu Matsushita (1810-1820).
The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost.Display Omitted
Keywords: Heme-copper oxidase; Ubiquinol oxidase; Cytochrome c oxidase; Heme synthase; Horizontal gene transfer; Acetic acid bacteria;

Photo-oxidation of tyrosine in a bio-engineered bacterioferritin ‘reaction centre’—A protein model for artificial photosynthesis by Kastoori Hingorani; Ron Pace; Spencer Whitney; James W. Murray; Paul Smith; Mun Hon Cheah; Tom Wydrzynski; Warwick Hillier (1821-1834).
The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates.Light-induced electron transfer from proximal tyrosine residues to the photo-oxidised ZnCe6 •+, in the modified BFR reconstituted with both ZnCe6 and MnII, is presented. Three site-specific tyrosine variants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in the engineered system. The results indicate that: presence of bound MnII is necessary to observe tyrosine oxidation in all BFR variants; Y45 the most important tyrosine as an immediate electron donor to the oxidised ZnCe6 •+ and that Y25 and Y58 are both redox-active in this system, but appear to function interchangebaly. High-resolution (2.1 Å) crystal structures of the tyrosine variants show that there are no mutation-induced effects on the overall 3-D structure of the protein. Small effects are observed in the Y45F variant. Here, the BFR-RC represents a protein model for artificial photosynthesis.
Keywords: Artificial photosynthesis; Protein engineering; Electron transfer; Tyrosine oxidation; Photosystem II; Bacterioferritin;

Efficiency of light harvesting in a photosynthetic bacterium adapted to different levels of light by Kõu Timpmann; Manoop Chenchiliyan; Erko Jalviste; John A. Timney; C. Neil Hunter; Arvi Freiberg (1835-1846).
In this study, we use the photosynthetic purple bacterium Rhodobacter sphaeroides to find out how the acclimation of photosynthetic apparatus to growth conditions influences the rates of energy migration toward the reaction center traps and the efficiency of charge separation at the reaction centers. To answer these questions we measured the spectral and picosecond kinetic fluorescence responses as a function of excitation intensity in membranes prepared from cells grown under different illumination conditions. A kinetic model analysis yielded the microscopic rate constants that characterize the energy transfer and trapping inside the photosynthetic unit as well as the dependence of exciton trapping efficiency on the ratio of the peripheral LH2 and core LH1 antenna complexes, and on the wavelength of the excitation light. A high quantum efficiency of trapping over 80% was observed in most cases, which decreased toward shorter excitation wavelengths within the near infrared absorption band. At a fixed excitation wavelength the efficiency declines with the LH2/LH1 ratio. From the perspective of the ecological habitat of the bacteria the higher population of peripheral antenna facilitates growth under dim light even though the energy trapping is slower in low light adapted membranes. The similar values for the trapping efficiencies in all samples imply a robust photosynthetic apparatus that functions effectively at a variety of light intensities.Display Omitted
Keywords: Photosynthesis; Photosynthetic unit; Optical spectroscopy; Light harvesting; Picosecond excitation energy transfer; Exciton;