BBA - Bioenergetics (v.1827, #10)
Editorial Board (i).
A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase by Victor W.T. Cheng; Quang M. Tran; Nasim Boroumand; Richard A. Rothery; Elena Maklashina; Gary Cecchini; Joel H. Weiner (1141-1147).
The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe–4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe–4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe–4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe–4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (> 100 mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.
Keywords: Complex II; Succinate dehydrogenase; Iron–sulfur cluster; Quinone binding site;
Molecular dynamics simulations reveal highly permeable oxygen exit channels shared with water uptake channels in photosystem II by Serguei Vassiliev; Tatiana Zaraiskaya; Doug Bruce (1148-1155).
Photosystem II (PSII) catalyzes the oxidation of water in the conversion of light energy into chemical energy in photosynthesis. Water delivery and oxygen removal from the oxygen evolving complex (OEC), buried deep within PSII, are critical requirements to facilitate the reaction and minimize reactive oxygen damage. It has often been assumed that water and oxygen travel through separate channels within PSII, as demonstrated in cytochrome c oxidase. This study describes all-atom molecular dynamics simulations of PSII designed to investigate channels by fully characterizing the distribution and permeation of both water and oxygen. Interestingly, most channels found in PSII were permeable to both oxygen and water, however individual channels exhibited different energetic barriers for the two solutes. Several routes for oxygen diffusion within PSII with low energy permeation barriers were found, ensuring its fast removal from the OEC. In contrast, all routes for water showed significant energy barriers, corresponding to a much slower permeation rate for water through PSII. Two major factors were responsible for this selectivity: (1) hydrogen bonds between water and channel amino acids, and (2) steric restraints. Our results reveal the presence of a shared network of channels in PSII optimized to both facilitate the quick removal of oxygen and effectively restrict the water supply to the OEC to help stabilize and protect it from small water soluble inhibitors.
Keywords: Photosystem II; Oxygen permeation; Molecular dynamics;
Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates by Ilka Siebels; Stefan Dröse (1156-1164).
The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.
Keywords: Mitochondria; Complex II; Succinate:ubiquinone oxidoreductase; Reactive oxygen species (ROS); Dicarboxylates; Atpenin A5;
Highly resolved proton matrix ENDOR of oriented photosystem II membranes in the S2 state by Hiroki Nagashima; Hiroyuki Mino (1165-1173).
Proton matrix ENDOR was performed to investigate the protons close to the manganese cluster in oriented samples of photosystem II (PS II). Eight pairs of ENDOR signals were detected in oriented PS II membranes. At an angle of θ = 0° between the membrane normal vector n and the external field H 0 , five pairs of ENDOR signals were exchangeable in D2O medium and three pairs were not exchangeable in D2O medium. The hyperfine splitting of 3.60 MHz at θ = 0° increased to 3.80 MHz at θ = 90°. The non-exchangeable signals with 1.73 MHz hyperfine splitting at θ = 0°, which were assigned to a proton in an amino acid residue, were not detected at θ = 90° in oriented PS II or in non-oriented PS II. Highly resolved spectra show that only limited numbers of protons were detected by CW-ENDOR spectra, although many protons were located near the CaMn4O5 cluster. The detected exchangeable protons were proposed to arise from the protons belonging to the water molecules, labeled W1-W4 in the 1.9 Å crystal structure, directly ligated to the CaMn4O5 cluster, and nearby amino-acid residue.Display Omitted
Keywords: Photosystem II; Oxygen evolving; Mn-cluster; EPR; ENDOR; Photosynthesis;
The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus by Alain Boussac; Kazumi Koyama; Miwa Sugiura (1174-1182).
Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II (PSII) complex. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues of PsbA, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this study, we found a new hemoprotein that is expressed when the T. elongatus genome has only the psbA2 gene for D1. This hemoprotein was found in both the non-membrane proteins and associated to the purified PsbA2-PSII core complex. This protein could be removed by the washing of PSII with Tris-washing or CaCl2-washing. From MALDI-TOF/TOF spectrometry, N-terminal sequencing and MALDI-MS/MS analysis upon tryptic digestion, the new hemoprotein was identified to be the tll0287 gene product with a molecular mass close to 19 kDa. Until now, tll0287 was registered as a gene encoding a hypothetical protein with an unknown function. From the amino acid sequence and the EPR spectrum the 5th and 6th axial ligands of the heme iron are the His145 and likely either the Tyr93, Tyr159 or Tyr165, respectively. From EPR, the heme containing Tll0287 protein associated to PsbA2-PSII corresponds to approximately 25% of the Cytc 550 content whereas, from SDS page analysis, the total amount of Tll0287 with and without the heme seems almost in a stoichiometric amount with PsbA2-PSII. Homologous genes to tll0287 are found in several cyanobacteria. Possible roles for Tll0287 are suggested.
Keywords: Photosystem II; PsbA; D1; Tll0287; Thermosynechococcus elongatus; Cyanobacterium;
Acetate in mixotrophic growth medium affects photosystem II in Chlamydomonas reinhardtii and protects against photoinhibition by Thomas Roach; Arezki Sedoud; Anja Krieger-Liszkay (1183-1190).
Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris–acetate–phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris–acetate–phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less 1O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of 1O2 originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (E m) of the quinone electron acceptors. Thermoluminescence indicated that the E m of the primary quinone acceptor (QA/QA −) of mixotrophic cells was stabilised while the E m of the secondary quinone acceptor (QB/QB −) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to 1O2 production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting photosystem II charge recombination events and photoinhibition.
Keywords: Acetate; Singlet oxygen; Photoinhibition; Thermoluminescence; Electron paramagnetic resonance spectroscopy; Chlamydomonas reinhardtii;
Mutational control of bioenergetics of bacterial reaction center probed by delayed fluorescence by Delphine Onidas; Gábor Sipka; Emese Asztalos; Péter Maróti (1191-1199).
The free energy gap between the metastable charge separated state P+QA − and the excited bacteriochlorophyll dimer P* was measured by delayed fluorescence of the dimer in mutant reaction center proteins of the photosynthetic bacterium Rhodobacter sphaeroides. The mutations were engineered both at the donor (L131L, M160L, M197F and M202H) and acceptor (M265I and M234E) sides. While the donor side mutations changed systematically the number of H-bonds to P, the acceptor side mutations modified the energetics of QA by altering the van-der-Waals and electronic interactions (M265IT) and H-bond network to the acidic cluster around QB (M234EH, M234EL, M234EA and M234ER). All mutants decreased the free energy gap of the wild type RC (~ 890 meV), i.e. destabilized the P+QA − charge pair by 60–110 meV at pH 8. Multiple modifications in the hydrogen bonding pattern to P resulted in systematic changes of the free energy gap. The destabilization showed no pH-dependence (M234 mutants) or slight increase (WT, donor-side mutants and M265IT above pH 8) with average slope of 10–15 meV/pH unit over the 6–10.5 pH range. In wild type and donor-side mutants, the free energy change of the charge separation consisted of mainly enthalpic term but the acceptor side mutants showed increased entropic (even above that of enthalpic) contributions. This could include softening the structure of the iron ligand (M234EH) and the QA binding pocket (M265IT) and/or increase of the multiplicity of the electron transfer of charge separation in the acceptor side upon mutation.
Keywords: Bacterial photosynthesis; Reaction center protein; H-bond network; Light-induced free energy changes; Charge recombination;
Completion of biosynthetic pathways for bacteriochlorophyll g in Heliobacterium modesticaldum: The C8-ethylidene group formation by Yusuke Tsukatani; Haruki Yamamoto; Tadashi Mizoguchi; Yuichi Fujita; Hitoshi Tamiaki (1200-1204).
Heliobacteria have the simplest photosynthetic apparatus, i.e., a type-I reaction center lacking a peripheral light-harvesting complex. Bacteriochlorophyll (BChl) g molecules are bound to the reaction center complex and work both as special-pair and antenna pigments. The C8-ethylidene group formation for BChl g is the last missing link in biosynthetic pathways for bacterial special-pair pigments, which include BChls a and b as well. Here, we report that chlorophyllide a oxidoreductase (COR) of Heliobacterium modesticaldum catalyzes the C8-ethylidene formation from 8-vinyl-chlorophyllide a, producing bacteriochlorophyllide g, the direct precursor for BChl g without the farnesyl tail. The finding led to plausible biosynthetic pathways for 81-hydroxy-chlorophyll a, a primary electron acceptor from the special pair in heliobacterial reaction centers. Proposed catalytic mechanisms on hydrogenation reaction of the ethylidene synthase-type CORs are also discussed.
Keywords: Bacteriochlorophyll; Chlorophyll; Chlorophyllide a oxidoreductase; Heliobacteria; Nitrogenase-like enzyme; Photosynthetic reaction center;
Quantitative calculation of the role of the Na+,K+-ATPase in thermogenesis by Ronald J. Clarke; Michelina Catauro; Helge H. Rasmussen; Hans-Jürgen Apell (1205-1212).
The Na+,K+-ATPase is accepted as an important source of heat generation (thermogenesis) in animals. Based on information gained on the kinetics of the enzyme's partial reactions we consider via computer simulation whether modifications to the function of the combined Na+,K+-ATPase/plasma membrane complex system could lead to an increased body temperature, either through the course of evolution or during an individual's lifespan. The enzyme's kinetics must be considered because it is the rate of heat generation which determines body temperature, not simply the amount of heat per enzymatic cycle. The results obtained indicate that a decrease in thermodynamic efficiency of the Na+,K+-ATPase, which could come about by Na+ substituting for K+ on the enzyme's extracellular face, could not account for increased thermogenesis. The only feasible mechanisms are an increase in the enzyme's expression level or an increase in its ion pumping activity. The major source of Na+,K+-ATPase-related thermogenesis (72% of heat production) is found to derive from passive Na+ diffusion into the cell, which counterbalances outward Na+ pumping to maintain a constant Na+ concentration gradient across the membrane. A simultaneous increase in both Na+,K+-ATPase activity and the membrane's passive Na+ permeability could promote a higher body temperature.
Keywords: Sodium pump; Heat generation; Albers–Post cycle; Transport pathways; Kinetic simulations; Power output;
High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system by Arnaldur Hall; Anna K. Larsen; Ladan Parhamifar; Kathrine D. Meyle; Lin-Ping Wu; S. Moein Moghimi (1213-1225).
Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25 kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in ‘broken mitochondria’ (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.
Keywords: Cell death; Cytochrome c oxidase; Electron transport system; Mitochondrial membrane potential; Mitochondrial uncoupling; Polyethylenimine;
Triplet–triplet energy transfer in fucoxanthin-chlorophyll protein from diatom Cyclotella meneghiniana: Insights into the structure of the complex by Marilena Di Valentin; Elena Meneghin; Laura Orian; Antonino Polimeno; Claudia Büchel; Enrico Salvadori; Christopher W.M. Kay; Donatella Carbonera (1226-1234).
Although the major light harvesting complexes of diatoms, called FCPs (fucoxanthin chlorophyll a/c binding proteins), are related to the cab proteins of higher plants, the structures of these light harvesting protein complexes are much less characterized. Here, a structural/functional model for the “core” of FCP, based on the sequence homology with LHCII, in which two fucoxanthins replace the central luteins and act as quenchers of the Chl a triplet states, is proposed. Combining the information obtained by time-resolved EPR spectroscopy on the triplet states populated under illumination, with quantum mechanical calculations, we discuss the chlorophyll triplet quenching in terms of the geometry of the chlorophyll–carotenoid pairs participating to the process. The results show that local structural rearrangements occur in FCP, with respect to LHCII, in the photoprotective site.
Keywords: Triplet state; FCP; Carotenoid; EPR; ODMR; Fucoxanthin;
Comparison of the physical characteristics of chlorosomes from three different phyla of green phototrophic bacteria by Peter G. Adams; Ashley J. Cadby; Benjamin Robinson; Yusuke Tsukatani; Marcus Tank; Jianzhong Wen; Robert E. Blankenship; Donald A. Bryant; C. Neil Hunter (1235-1244).
Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered “Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum” were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133 × 57 × 36 nm; 141,000 nm3 volume), compared with chlorosomes from Cfx. aurantiacus (120 × 44 × 30 nm; 84,000 nm3) and Ca. Cab. thermophilum (99 × 40 × 31 nm; 65,000 nm3). Reflecting the contributions of thousands of pigment–pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000–2000 pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30 nm wide and 6 nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.
Keywords: Bacterial photosynthesis; Light harvesting; Chlorosome; Atomic force microscopy; Fluorescence microscopy;
Mitochondrial glutamate carriers from Drosophila melanogaster: Biochemical, evolutionary and modeling studies by Paola Lunetti; Anna Rita Cappello; René Massimiliano Marsano; Ciro Leonardo Pierri; Chiara Carrisi; Emanuela Martello; Corrado Caggese; Vincenza Dolce; Loredana Capobianco (1245-1255).
The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.
Keywords: Drosophila melanogaster; CG18347 and CG12201; Proteomics; Glutamate carrier; Phylogenesis;