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BBA - Bioenergetics (v.1817, #2)
Electrogenic events upon photolysis of CO from fully reduced cytochrome c oxidase by Marko Rintanen; Ilya Belevich; Michael I. Verkhovsky (pp. 269-275).
CO photolysis from fully reduced Paracoccus denitrificans aa3-type cytochrome c oxidase in the absence of O2 was studied by time-resolved potential electrometry. Surprisingly, photo dissociation of the uncharged carbon monoxide results in generation of a small-amplitude electric potential with the same sign as the physiological charge separation during activity. The number of electrogenic events after CO photolysis depends on the state of the enzyme. CO photolysis following immediately after activation by an enzymatic turnover, showed a two-component potential development. A fast (~1.5μs) phase was followed by slower potential generation with a time constant varying from 8μs at pH 7 to 250μs at pH 10. The amplitude of the fast phase was independent of the time of incubation after enzyme activation, whereas the slower phase vanished with a time constant of ~25min. CO photolysis from enzyme that had not undergone a prior single turnover showed the fast phase, but the amplitude of the slow phase was reduced to 10–30%. The amplitude of the fast phase corresponds to charge movement of 0.83Å perpendicular to the membrane dielectric, and is independent of the time after enzyme activation. Thus it can be used as an internal ruler for normalization of the electrogenic responses of C cO. The slow phase was absent in the K354M mutant with a blocked proton-conducting K channel. We propose that CO photolysis increases the pK of the K354 residue, which results in its partial protonation, and generation of electric potential.► CO photolysis from fully reduced cytochrome c oxidase after turnover. ► Two phases of potential generation. ► Fast phase has constant amplitude and time constant in a pH range of 7 to 10. ► Slower phase has pH dependent time constant and amplitude. ► Slower phase is related to K channel.
Keywords: Abbreviations; C; c; O; type; aa; 3; cytochrome; c; oxidase; DM; n; -dodecyl-β-; d; maltoside; E; h; ambient redox potential; E; m; midpoint redox potentialPotential generation; Proton transfer; Potential electrometry; Molecular bioenergetics
Biogenesis of the cytochrome bc1 complex and role of assembly factors by Pamela M. Smith; Jennifer L. Fox; Dennis R. Winge (pp. 276-286).
The cytochrome bc1 complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc1 complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc1 complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc1 complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc1 complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc1 complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.► Biogenesis of bc1 complexes occurs as a modular assembly pathway. ► Stable assembly intermediates accumulate in mutants stalled in biogenesis. ► Recently identified assembly factors function in the formation of assembly intermediates.
Keywords: Cytochrome; bc; 1; complex; Rieske Fe/S protein; Bcs1; Mzm1; Cyt1
Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II by Shigeru Itoh; Takashi Kozuki; Koji Nishida; Yoshimasa Fukushima; Hisanori Yamakawa; Ildikó Domonkos; Laczko-Dobos Hajnalka Laczkó-Dobos; Mihály Kis; Bettina Ughy; Zoltán Gombos (pp. 287-297).
Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of QB to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PGL) enhances the affinity for BQ that inactivates QB. 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of QB during 10days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PGH) displaces QB directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14days decreased the chlorophyll/PBS ratio to about 1/4, florescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PGH, PGL and other PG molecules on PSII structure are discussed.► Phosphatidylglycerol was depleted from cyanobacterial pgsA-gene-inactivated cells. ► Two PG molecules regulate activities of QB and oxygen evolution differently. ► Loss of high-affinity PGH inhibits QB directly and suppress intrinsic O2 evolution. ► Loss of low-affinity PGL enhances PSII affinity to BQ that inactivates QB. ► Binding sites of PGH and PGL in PS II structure are estimated.
Keywords: Abbreviations; APC; allophycocyanin; BQ; p; -benzoquinone; Chl; chlorophyll; D; (BQ); and D; 2(int); decay of fluorescence yield measured with and without BQ; DCMU; 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; DGDG; digalactosyldiacylglycerol; MGDG; monogalactosyldiacylglycerol; O; 2(BQ); and O; 2(int); oxygen evolution activity with and without BQ, respectively; PAM; pulse amplidue modulation; PC; phycocyanin; PG; phosphatidylglycerol; PG; H; and PG; L; PG bound to the high- and low-affinity sites that regulate the Q; B; function, respectively; PG1-5; PG molecules identified in the structure of PS II; PS; photosystem; Q; A; and Q; B; first and second electron acceptor plastoquinoneof PSII; RC; reaction center; SQDG; sulfoquinovosyldiacyglycerol; WT; wild typeChlorophyll fluorescence; Lipid; Oxygen evolution; Phosphatidylglycerol; Photosystem II assembly; Q; B; plastoquinone
Generation, characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides by Ting Su; Lothar Esser; Di Xia; Chang-An Yu; Linda Yu (pp. 298-305).
Cytochrome bc1 complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c2) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc1 complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c1 with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV ( c1-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5Å resolution.► We have generated R. sphaeroides mutant bc1 complex c1-14Gly-IV-6His. ► The purified mutant complex shows more structural stability. ► Crystals (5.5Å) containing four subunits were obtained.
Keywords: Abbreviations; ISP; iron–sulfur protein; Q; 0; C; 10; BrH; 2; 2,3-dimethoxy-5- methyl-6-(10-bromodecyl)-1,4-benzoquinol; DM; dodecylmaltoside; OG; n-; octyl; -beta-; d; -glucopyranoside; QoC10BrH; 2; 2,3-dimethoxy-5-methyl-(10-bromodecyl)1,4-benzoquinol; MCLA; 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one hydrocholorideSupernumerary subunit; Bacterial cytochrome; bc; 1; complex; Rhodobacter sphaeroides; Fusion protein
Stabilization and modulation of the phycobilisome by calcium in the calciphilic freshwater red alga Bangia atropurpurea by Makio Yokono; Hiroko Uchida; Yuzuru Suzawa; Seiji Akiomoto; Akio Murakami (pp. 306-311).
The bangiophycean filamentous red alga Bangia atropurpurea is distributed in freshwater habitats such as littoral and splash zones of lakes or rapid currents distant from the sea. In these habitats, the distribution and growth of this alga appear to be related to hard water rich in calcium ions. To characterize the eco-physiological properties of this calciphilic red alga, we examined the effects of long-term and short-term Ca2+ depletion on photosynthetic growth of the thallus and on the phycobilisome. Long-term culture experiments suggested that higher Ca2+ concentrations (>50mgL−1) were required to sustain thallus growth and pigmentation of cells. In short-term Ca2+-depletion treatments, fluorescence derived from phycoerythrin (PE) fluctuated, although the absorption spectra of the thalli did not change. After 30min of Ca2+ depletion, the fluorescence lifetime of PE became markedly longer, indicating that the energy transfer from PE to phycocyanin (PC) was suppressed. The fluorescence lifetime of PE returned to its original value within a short time after 4h of Ca2+ depletion, however, energy transfer from PE to PC was still suppressed. This suggested that the excitation energy absorbed by PE was quenched during prolonged Ca2+ depletion. The efficient energy transfer from PC and allophycocyanin were unchanged during these treatments.►The freshwater red alga Bangia atropurpurea is distributed in hard water habitats. ►Higher concentrations of calcium were essential for photosynthetic growth of B. atropurpurea. ►Calcium depletion treatments resulted in phycoerythrin decoupling and quenching.
Keywords: Abbreviations; PBS; phycobilisome; PE; phycoerythrin; PC; phycocyanin; APC; allophycocyanin; TRFS; time-resolved fluorescence spectra Bangia atropurpurea; Calcium; Phycobilisome; Energy transfer; Red algae
LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1 by Patsi Jukka Pätsi; Pilvi Maliniemi; Salla Pakanen; Reetta Hinttala; Johanna Uusimaa; Kari Majamaa; Nystrom Thomas Nyström; Marko Kervinen; Ilmo E. Hassinen (pp. 312-318).
Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K m for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.► ND1 subunit is adjacent to the ubiquinone binding site of complex I. ► Involvement of E. coli NuoH-E36, the homolog of ND1-E24, in quinone binding is shown. ► ND1-E24 lies in the ubiquinone binding domain delineated by PSST and 49kDa subunits. ► NUOH-E36 mutation effects corroborate the m.3376G>A pathogenicity in LHON/MELAS. ► m.3376G>A combines enzymic consequences of separate LHON and MELAS mutations.
Keywords: Abbreviations; d-NADH; deamino-NADH; DB; decylubiquinone; HAR; hexaammineruthenium; LHON; Leber hereditary optic neuropathy; MELAS; mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes syndrome; mtDNA; mitochondrial DNA; NDH-1; bacterial proton pumping NADH-ubiquinone oxidoreductase; NDH-2; bacterial non-proton pumping NADH-ubiquinone oxidoreductase; Q1; ubiquinone-1; Q2; ubiquinone-2; UQ; ubiquinone; VNA; N-vanillylnonanamide Escherichia coli; Leber hereditary optic neuropathy; MELAS; Mitochondria; NADH-ubiquinone oxidoreductase; Ubiquinone
Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress by Eyal Tamary; Vladimir Kiss; Reinat Nevo; Zach Adam; Bernat Gábor Bernát; Sascha Rexroth; Rogner Matthias Rögner; Ziv Reich (pp. 319-327).
Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.► High-light induces energetic decoupling of PBSs from the reaction centers. ► Energetic decoupling is accompanied by detachment/disassembly of the PBSs. ► The above processes are likely induced by thermal transients within the PBS. ► Energetic decoupling of PBSs may serve a photoprotective role under stress conditions.
Keywords: Abbreviations; APC; allophycocyanin; Chl; chlorophyll; NPQ; non-photochemical quenching; OCP; orange carotenoid protein; PBSs; phycobilisomes; PC; phycocyanin; PSI; photosystem I; PSII; photosystem IICyanobacteria; Energetic decoupling; Energy transfer; FRAP; Photoprotection; Phycobilisomes
Kinetics of phyllosemiquinone oxidation in the Photosystem I reaction centre of Acaryochloris marina by Stefano Santabarbara; Benjamin Bailleul; Kevin Redding; James Barber; Fabrice Rappaport; Alison Telfer (pp. 328-335).
Light-induced electron transfer reactions in the chlorophyll a/ d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5–30ns and 200–300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.► The oxidation kinetics of secondary intermediates in PS I of A. marina are biphasic. ► The decay is characterised by lifetimes of 88±6ns and 345±10ns. ► Both these lifetimes are slower than in typical Chl a-binding PS I reaction centres. ► Possible mechanism of electron transfer is discussed.
Keywords: Abbreviations; Chl; chlorophyll; DCMU; 3-3,4 dichlorophenyl-1,1-dimethylurea; FCCP; carbonyl-cyanide-; p; -trifluoromethoxy-phenylhydrazone; PhQ; phylloquinone; PS (I/II); Photosystem (I/II); RC; reaction centres; P; (+); or P; 700/740; (+); PS I electron donor (cation)Bidirectional electron transfer; Photosystem I; Chlorophyll; d; Radical pair; Phylloquinone; Acaryochloris marina
Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex by Lucy I. Crouch; Michael R. Jones (pp. 336-352).
In well-characterised species of the Rhodobacter ( Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.Display Omitted► Dimeric RC–LH1 complexes were be isolated from Rba. azotoformans and Rba. changlensis. ► RC–LH1 dimers could not be isolated from Rba. capsulatus or Rba. vinaykumarii. ► Chimeric RC–LH1 complexes could be isolated as monomers but not as dimers. ► Whether RC–LH1 dimers can be isolated is determined by both PufX and LH1 proteins. ► The findings tease apart different functions of PufX in different Rhodobacter species.
Keywords: Abbreviations; AFM; atomic force microscopy; A; LH1; /A; RC; ratio of LH1 absorbance to RC absorbance; BChl; bacteriochlorophyll; DM; decyl β-; d; -maltoside; DDM; n-dodecyl-β-; d; -maltoside; DHPC; 1,2-diheptanoyl-; sn; -glycero-3-phosphocholine; EM; electron microscopy; LDAO; N,N-dimethyldodecylamine N-oxide; LH1; light-harvesting 1 pigment protein; LH2; light-harvesting 2 pigment protein; NMR; nuclear magnetic resonance; OG; octyl β-; d; -glucoside; PDB; Protein Data Bank; PMS; phenazine methosulphate; RC; reaction centre; Rba; Rhodobacter; Rps; RhodopseudomonasPhotosynthesis; Rhodobacter sphaeroides; Reaction centre; Light harvesting; PufX; LH1 antenna
During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway by Sergio Guerrero-Castillo; Alfredo Cabrera-Orefice; Vazquez-Acevedo Miriam Vázquez-Acevedo; Gonzalez-Halphen Diego González-Halphen; Salvador Uribe-Carvajal (pp. 353-362).
In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e–AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.► Interactions of orthodox and alternative respiratory enzymes are dynamic. ► Association/dissociation of respiratory enzymes modulates electron pathways. ► Pathways condition coupling and control ROS production. ► In the stat-phase an uncoupled alternative pathway is activated. ► In the stat-phase, NDH2e increases while complex IV decreases.
Keywords: Abbreviations; NDH2e; alternative external NADH dehydrogenase; AOX; alternative oxidase; ΔΨ; transmembrane potential; CCCP; carbonylcyanide-3-chlorophenylhydrazone; ROS; reactive oxygen species Yarrowia lipolytica; Mitochondrion; Branched respiratory chain; Alternative NADH dehydrogenase; Physiological uncoupling; Reactive oxygen species, ROS
The effects of idebenone on mitochondrial bioenergetics by Valentina Giorgio; Valeria Petronilli; Anna Ghelli; Valerio Carelli; Michela Rugolo; Giorgio Lenaz; Paolo Bernardi (pp. 363-369).
We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at aminoacid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 [Haefeli et al. (2011) PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.► The mitochondrial effects of idebenone in living cells are poorly characterized. ► Idebenone can be toxic or beneficial depending on its oxidation-reduction state. ► Idebenone is toxic through opening of the permeability transition pore. ► Idebenol stimulates respiration and ATP synthesis in cells lacking complex I. ► Short-chain quinones can be developed for the treatment of complex I defects.
Keywords: Abbreviations; CRC; calcium retention capacity; Cs; cyclosporin; DMEM; Dulbecco's modified Eagle's medium; Δψ; m; mitochondrial membrane potential difference; DTT; dithiothreitol; FCCP; carbonylcyanide-; p; -trifluoromethoxyphenyl hydrazone; LHON; Leber's hereditary optic neuropathy; MELAS; mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; MOPS; 4-morpholinepropanesulfonic acid; NQO1; NAD(P)H:quinone oxidoreductase 1; OCR; oxygen consumption rate; PTP; permeability transition pore; ROS; reactive oxygen species; TMRM; tetramethylrhodamine methyl esterMitochondria; Idebenone; Permeability transition; Electron transfer; ATP synthesis
Physiology and global gene expression of a Corynebacterium glutamicum ΔF1FO-ATP synthase mutant devoid of oxidative phosphorylation by Abigail Koch-Koerfges; Armin Kabus; Ines Ochrombel; Kay Marin; Michael Bott (pp. 370-380).
A mutant of Corynebacterium glutamicum ATCC 13032 with a deletion of the atpBEFHAGDC genes encoding F1FO-ATP synthase was characterized. Whereas no growth was observed with acetate as sole carbon source, the ΔF1FO mutant reached 47% of the growth rate and 65% of the biomass of the wild type during shake-flask cultivation in glucose minimal medium. Initially, the mutant strain showed a strongly increased glucose uptake rate accompanied by a high oxygen consumption rate and pyruvate secretion into the medium. When oxygen became limiting, the glucose consumption rate was reduced below that of the wild type and pyruvate was consumed again. The ΔF1FO mutant had increased levels of b- and d-type cytochromes and a significantly increased proton motive force. Transcription of genes involved in central carbon metabolism was essentially unchanged, whereas genes for cytochrome bd oxidase, pyruvate:quinone oxidoreductase, oxidative stress response, and others showed increased mRNA levels. On the other hand, genes for amino acid biosynthesis and ribosomal proteins as well as many genes involved in transport displayed decreased mRNA levels. Several of the transcriptional changes were reflected at the protein level, but there were also discrepancies between the mRNA and protein levels suggesting some kind of posttranscriptional regulation. The results prove for the first time that F1FO-ATP synthase and oxidative phosphorylation are in general not essential for growth of C. glutamicum.► We describe a C. glutamicum mutant with a deletion of the F1FO-ATP synthase genes. ► Growth of DF1FO mutant proves that oxidative phosphorylation is dispensable. ► Under O2 excess DF1FO shows increased glucose consumption, respiration and DΨ. ► Transcriptomics reveals ~300 genes with ≥2-fold altered mRNA levels in DF1FO. ► Proteomics reveals ~30 proteins with ≥1.5-fold altered protein levels in in DF1FO.
Keywords: F1FO-ATP synthase; Oxidative phosphorylation; atpBEFHAGDC; deletion; Corynebacterium glutamicum; Proton motive force; Gene expression
Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase by Sascha Rexroth; Ansgar Poetsch; Rogner Matthias Rögner; Andrea Hamann; Alexandra Werner; Heinz D. Osiewacz; Schafer Eva R. Schäfer; Holger Seelert; Norbert A. Dencher (pp. 381-387).
The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia.In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.► Selective oxidation of specific tryptophan residue. ► Identification and quantification of oxidation intermediates. ► Molecular mechanism for selective targeting.
Keywords: Abbreviations; Desferal; Deferoxamine N′-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide; FRTA; Free radical theory of ageing; Kyn; kynurenine; MFRTA; mitochondrial free radical theory of ageing; MS; mass spectrometry; NFK; N; -formyl-kynurenine; ROS; reactive oxygen species; SRM; single reaction monitoring; TRP-OH; hydroxy-tryptophanATP synthase; Oxidative stress; Podospora anserina; Aging; Posttranslational modification