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BBA - Bioenergetics (v.1817, #9)

Editorial Board (pp. i).

RNA helicase, CrhR is indispensable for the energy redistribution and the regulation of photosystem stoichiometry at low temperature in Synechocystis sp. PCC6803 by Kodru Sireesha; Balaga Radharani; Pilla Sankara Krishna; Nellaepalli Sreedhar; Rajagopal Subramanyam; Prasanna Mohanty; Jogadhenu S.S. Prakash (pp. 1525-1536).

We investigated the role of a cold-inducible and redox-regulated RNA helicase, CrhR, in the energy redistribution and adjustment of stoichiometry between photosystem I (PSI) and photosystem II (PSII), at low temperature in Synechocystis sp. PCC 6803. The results suggest that during low temperature incubation, i.e., when cells are shifted from 34°C to 24°C, wild type cells exhibited light-induced state transitions, whereas the mutant deficient in CrhR failed to perform the same. At low temperature, wild type cells maintained the plastoquinone (PQ) pool in the reduced state due to enhanced respiratory electron flow to the PQ pool, whereas in ∆ crhR mutant cells the PQ pool was in the oxidized state. Wild type cells were in state 2 and ∆ crhR cells were locked in state 1 at low temperature. In both wild type and ∆ crhR cells, a fraction of PSI trimers were changed to PSI monomers. However, in ∆ crhR cells, the PSI trimer content was significantly decreased. Expression of photosystem I genes, especially the psaA and psaB, was strongly down-regulated due to oxidation of downstream components of PQ in ∆ crhR cells at low temperature. We demonstrated that changes in the low temperature-induced energy redistribution and regulation of photosystem stoichiometry are acclimatization responses exerted by Synechocystis cells, essentially regulated by the RNA helicase, CrhR, at low temperature.► Redox of PQ-downstream e^- carriers signals psaAB expression at low temperature (LT). ► Synechocystis acclimatize to LT by controlling PS stoichiometry & State transitions. ► Δ crhR unable to maintain PQ redox state & failed to operate state transitions at LT. ► In Δ crhR, oxidized state of e- carriers leads to PSI genes down regulation at LT. ► CrhR is essential for state transitions & PS stoichiometry regulation at LT.

Keywords: Abbreviations; PBQ; p-benzoquinone; PQ; plastoquinone; PS; photosystem; WT; wild type; PAM; pulse amplitude modulator Synechocystis; sp. PCC6803; Low temperature; Photosystem stoichiometry; Redox regulation; ∆; crhR; mutant

Induction of the mitochondrial permeability transition (MPT) by micromolar iron: Liberation of calcium is more important than NAD(P)H oxidation by Gall Juraj Gáll; Skrha Jan Škrha Jr.; Richard Buchal; Sedlackova Eva Sedláčková; Verebova Karina Verébová; Platenik Jan Pláteník (pp. 1537-1549).

The mitochondrial permeability transition (MPT) plays an important role in cell death. The MPT is triggered by calcium and promoted by oxidative stress, which is often catalyzed by iron. We investigated the induction of the MPT by physiological concentrations of iron. Isolated rat liver mitochondria were initially stabilized with EDTA and bovine serum albumin and energized by succinate or malate/pyruvate. The MPT was induced by 20μM calcium or ferrous chloride. We measured mitochondrial swelling, the inner membrane potential, NAD(P)H oxidation, iron and calcium in the recording medium. Iron effectively triggered the MPT; this effect differed from non-specific oxidative damage and required some residual EDTA in the recording medium. Evidence in the literature suggested two mechanisms of action for the iron: NAD(P)H oxidation due to loading of the mitochondrial antioxidant defense systems and uptake of iron to the mitochondrial matrix via a calcium uniporter. Both of these events occurred in our experiments but were only marginally involved in the MPT induced by iron. The primary mechanism observed in our experiments was the displacement of adventitious/endogenous calcium from the residual EDTA by iron. Although artificially created, this interplay between iron and calcium can well reflect conditions in vivo and could be considered as an important mechanism of iron toxicity in the cells.► 20μM Fe(II) induces mitochondrial permeability transition in isolated mitochondria. ► Iron causes NAD(P)H oxidation but this is only marginally involved in MPT induction. ► Small fraction of added iron was imported by calcium uniporter. ► MPT propagation continues through calcium release/reuptake. ► The effect of iron depends on its ability to displace calcium from residual EDTA.

Keywords: Mitochondrial permeability transition; Oxidative stress; Iron toxicity; Calcium signaling; Chelators

Tightly-bound ubiquinone in the Escherichia coli respiratory Complex I by Michael Verkhovsky; Dmitry A. Bloch; Marina Verkhovskaya (pp. 1550-1556).

NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe–S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270–280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50–+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials ( E_m ≤−200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.Display Omitted► Purified Complex I from E. coli contains bound ubiquinone. ► The fast events upon Complex reduction were followed optically. ► The results indicate fast redox transition of the bound ubiquinone. ► Midpoint redox potential of the bound ubiquinone is lower than –200mV.

Keywords: Abbreviations; BTP; 1,3-bis(tris(hydroxymethyl)methylamino)propane; DDM; n-dodecyl β-; d; -maltopyranoside; DQ; decylubiquinone; E; _h; ambient redox potential; E; _m; midpoint redox potential; HAR; hexaammineruthenium (III) chloride; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MOPS; 3-(N-morpholino)propanesulfonic acid; NHE; normal hydrogen electrode; OTTLE; optically transparent, thin layer electrode; Q1; ubiquinone-1; TMH; transmembrane helix; UQ; ubiquinone; UQH; ₂; ubiquinol; WE; working electrode; τ; time constantComplex I; Ubiquinone; Binding site; Stopped-flow; Redox titration

The harmful alga Aureococcus anophagefferens utilizes 19′-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities by Meriem Alami; Dusan Lazar; Beverley R. Green (pp. 1557-1564).

Aureococcus anophagefferens is a picoplanktonic microalga that is very well adapted to growth at low nutrient and low light levels, causing devastating blooms (“brown tides”) in estuarine waters. To study the factors involved in long-term acclimation to different light intensities, cells were acclimated for a number of generations to growth under low light (20μmolphotonsm−²s−¹), medium light (60 or 90μmolphotonsm−²s−¹) and high light (200μmolphotonsm−²s−¹), and were analyzed for their contents of xanthophyll cycle carotenoids (the D pool), fucoxanthin and its derivatives (the F pool), Chls c₂ and c₃, and fucoxanthin Chl a/c polypeptides (FCPs). Higher growth light intensities resulted in increased steady state levels of both diadinoxanthin and diatoxanthin. However, it also resulted in the conversion of a significant fraction of fucoxanthin to 19′-butanoyloxyfucoxanthin without a change in the total F pool. The increase in 19′-butanoyloxyfucoxanthin was paralleled by a decrease in the effective antenna size, determined from the slope of the change in F₀ as a function of increasing light intensity. Transfer of acclimated cultures to a higher light intensity showed that the conversion of fucoxanthin to its derivative was a relatively slow process (time-frame of hours). We suggest the replacement of fucoxanthin with the bulkier 19′-butanoyloxyfucoxanthin results in a decrease in the light-harvesting efficiency of the FCP antenna and is part of the long-term acclimative response to growth at higher light intensities.► We studied the acclimation of the "brown tide" alga Aureococcus anophagefferens to high light. ► Its acclimation processes utilize two different carotenoid-based strategies. ► One involves the xanthophyll cycle carotenoids; the other involves fucoxanthin derivatives. ► Up to 50% of fucoxanthin is converted to 19'-butanoyloxyfucoxanthin during growth at high light. ► Hypothesis: This is a photoprotective conversion which decreases energy transfer efficiency.

Keywords: Abbreviations; Chl; chlorophyll; Dd; diadinoxanthin; Dt; diatoxanthin; Fx; fucoxanthin; 19′-BFx; 19′-butanoyloxyfucoxanthin; LHC; light-harvesting complex; FCP; fucoxanthin Chl; a/c; protein; PAR; photosynthetically active radiation.Acclimation; Chlorophyll; a/c; protein; Fucoxanthin; 19′-butanoyloxyfucoxanthin; High light stress; Xanthophyll cycle

Evolution and diversification of Group 1 [NiFe] hydrogenases. Is there a phylogenetic marker for O₂-tolerance? by Maria-Eirini Pandelia; Wolfgang Lubitz; Wolfgang Nitschke (pp. 1565-1575).

Group 1 hydrogenases are periplasmic enzymes and are thus strongly affected by the “outside world” the cell experiences. This exposure has brought about an extensive heterogeneity in their cofactors and redox partners. Whereas in their majority they are very O₂-sensitive, several enzymes of this group have been recently reported to be O₂-tolerant. Structural and biochemical studies have shown that this O₂-tolerance is conferred by the presence of an unusual iron–sulfur cofactor with supernumerary cysteine ligation (6 instead of 4 Cys, hence called ‘6C cluster’). This atypical cluster coordination affords redox plasticity (i.e. two-redox transitions), unprecedented for this type of cofactors and likely involved in resistance to O₂. Genomic screening and phylogenetic tree reconstruction revealed that 6C hydrogenases form a monophyletic clade and are unexpectedly widespread among bacteria. However, several other well-defined clades are observed, which indicate early diversification of the enzyme into different subfamilies. The various idiosyncrasies thereof are shown to comply with a very simple rule: phylogenetic grouping of hydrogenases directly correlates with their specific functions and hence biochemical characteristics. The observed variability results from gene duplication, gene shuffling and subsequent adaptation of the diversified enzymes to specific environments. An important factor for this diversification seems to have been the emergence of molecular oxygen. Hydrogenases appear to have dealt with oxidative stress in various ways, the most successful of which, however, was the innovation of the 6C-cluster conferring pronounced O₂-tolerance to the parent enzymes.► O₂-tolerant hydrogenases harbor a novel [FeS] cofactor with 6 cysteine (6C) coordination. ► Through genomic screenings we map the 6C trait onto the most recent phylogenetic tree. ► Structural/functional characteristics correlate with the planet's geochemical history. ► 6C hydrogenases are omnipresent in recent microaerophilic and aerobic bacterial phyla. ► A link between 6C enzymes and increasing oxidative atmospheric conditions is proposed.

Keywords: Abbreviations; DMK; n; demethylmenaquinone; n; , where; n; refers to the number of the prenyl units present in the side-chain; UQ; n; ubiquinone; n; , where; n; refers to the number of the prenyl units present in the side-chain; MK; menaquinone; Hdr; heterodisulfide reductase; Dsr; dissimilatory sulfate reductase; CoB; coenzyme B (full chemical name 7-mercaptoheptanoylthreoninephosphate); CoM; coenzyme M is the anion with the formula HSCH; ₂; CH; ₂; SO; ₃; ⁻[NiFe] hydrogenases; Phylogenetic tree; O; ₂; -tolerance; Bioenergetics; Iron–sulfur cluster

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum by Dariusz M. Niedzwiedzki; David Bina; Nichola Picken; Suvi Honkanen; Robert E. Blankenship; Dewey Holten; Richard J. Cogdell (pp. 1576-1587).

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.► Femtosecond time-resolved spectroscopy of LH2 from Alc. vinosum. ► Carotenoid-to-bacteriochlorophyll a energy transfer. ► Femtosecond time-resolved spectroscopy of carotenoids. ► Explanation of mechanism of splitting of B800 bacteriochlorophyll a band in LH2. ► 77K spectroscopy.

Keywords: Time-resolved spectroscopy; Photochemistry of carotenoids; Light harvesting complex 2; Bacteriochlorophyll; a; Energy transfer

The stability and activity of respiratory Complex II is cardiolipin-dependent by Christine T. Schwall; Victoria L. Greenwood; Nathan N. Alder (pp. 1588-1596).

Respiratory Complex II of the mitochondrial inner membrane serves as a link between the tricarboxylic acid cycle and the electron transport chain. Complex II dysfunction has been implicated in a wide range of heritable mitochondrial diseases, including cancer, by a mechanism that likely involves the production of reactive oxygen species (ROS). Using Complex II enzymes reconstituted into nanoscale lipid bilayers (nanodiscs) with varying lipid composition, we demonstrate for the first time that the phospholipid environment, specifically the presence of cardiolipin, is critical for the assembly and enzymatic activity of the complex, as well as in the curtailment of ROS production.► We have reconstituted functional Respiratory Complex II into phospholipid nanodiscs. ► Cardiolipin is essential for the stability and enzymatic activity of Complex II. ► The presence of cardiolipin curtails the production of superoxide by Complex II.

Keywords: Abbreviations; CL; cardiolipin; DB; decylubiquinone; DCPIP; dichlorophenolindophenol; DDM; dodecyl-β-D-maltoside; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; EDTA; ethylenediaminetetraacetic acid; IM; inner membrane; MSP; membrane scaffolding protein; ND; nanodisc; OXPHOS; oxidative phosphorylation; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-ethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; ROS; reactive oxygen species; SDH; succinate dehydrogenase; SQR; succinate:ubiquinone oxidoreductase; TCA; tricarboxylic acid; TTFA; thenoyltrifluoroacetone; Q; ubiquinone; QH; ₂; ubiquinolComplex II; Nanodisc; Cardiolipin; ROS

Molecular mechanism for the selective impairment of cancer mitochondrial function by a mitochondrially targeted vitamin E analogue by Rodriguez-Enriquez Sara Rodríguez-Enríquez; Hernandez-Esquivel Luz Hernández-Esquivel; Marin-Hernandez Alvaro Marín-Hernández; Lan-Feng Dong; Emmanuel T. Akporiaye; Jiri Neuzil; Stephen J. Ralph; Moreno-Sanchez Rafael Moreno-Sánchez (pp. 1597-1607).

The effects of α-tocopheryl succinate (α-TOS), α-tocopheryl acetyl ether (α-TEA) and triphenylphosphonium-tagged vitamin E succinate (mitochondrially targeted vitamin E succinate; MitoVES) on energy-related mitochondrial functions were determined in mitochondria isolated from AS-30D hepatoma and rat liver, bovine heart sub-mitochondrial particles (SMPs), and in rodent and human carcinoma cell lines and rat hepatocytes. In isolated mitochondria, MitoVES stimulated basal respiration and ATP hydrolysis, but inhibited net state 3 (ADP-stimulated) respiration and Ca2⁺ uptake, by collapsing the membrane potential at low doses (1–10μM). Uncoupled mitochondrial respiration and basal respiration of SMPs were inhibited by the three drugs at concentrations at least one order of magnitude higher and with different efficacy: MitoVES>α-TEA>α-TOS. At high doses (>10μM), the respiratory complex II (CII) was the most sensitive MitoVES target. Acting as an uncoupler at low doses, this agent stimulated total O₂ uptake, collapsed ∆ ψ_m, inhibited oxidative phosphorylation and induced ATP depletion in rodent and human cancer cells more potently than in normal rat hepatocytes. These findings revealed that in situ tumor mitochondria are preferred targets of the drug, indicating its clinical relevance.Display Omitted► The TPP⁺-tagged vitamin E succinate (MitoVES) affected mitochondrial functions. ► MitoVES stimulated basal respiration and ATP hydrolysis in mitochondria. ► MitoVES inhibited OxPhos and Ca2⁺ uptake by collapsing the membrane potential in mitochondria. ► The respiratory complex II was the most sensitive MitoVES target. ► MitoVes potently inhibited OxPhos and induced ATP depletion in rodent and human cancer cells.

Keywords: Abbreviations; CII; respiratory complex II; MitoVES; triphenylphosphonium tagged vitamin E succinate; mtDNA; mitochondrial DNA; 2-OG; 2-oxoglutarate; OxPhos; oxidative phosphorylation; Pyr/Mal; pyruvate; +; malate; RLM; rat liver mitochondria; SDH; succinate dehydrogenase; SMPs; sub-mitochondrial particles; α-TEA; α-tocopheryl acetyl ether; α-TOS; α-tocopheryl succinate; TPP; ⁺; triphenylphosphonium; VE; vitamin E; TTFA; thenoyltrifluoroacetoneMitochondria; Respiratory complex II; Vitamin E analogue; Uncoupling; Tumor cells

NPQ activation reduces chlorophyll triplet state formation in the moss Physcomitrella patens by Donatella Carbonera; Caterina Gerotto; Bianca Posocco; Giorgio Mario Giacometti; Tomas Morosinotto (pp. 1608-1615).

Plants live in variable environments in which light intensity can rapidly change, from limiting to excess conditions. Non-photochemical quenching (NPQ) is a regulatory mechanism which protects plants from oxidative stress by dissipating excess Chl singlet excitation. In this work, the physiological role of NPQ was assessed by monitoring its influence on the population of the direct source of light excess damage, i.e., Chl triplets (³Chl*).³Chl* formation was evaluated in vivo, with the moss Physcomitrella patens, by exploiting the high sensitivity of fluorescence-detected magnetic resonance (FDMR). A dark adapted sample was compared with a pre-illuminated sample in which NPQ was activated, the latter showing a strong reduction in³Chl* yield. In line with this result, mutants unable to activate NPQ showed only a minor effect in³Chl* yield upon pre-illumination.The decrease in³Chl* yield is equally experienced by all the Chl pools associated with PSII, suggesting that NPQ is effective in protecting both the core and the peripheral antenna complexes. Moreover, the FDMR results show that the structural reorganization in the photosynthetic apparatus, required by NPQ, does not lead to the formation of new³Chl* traps in the LHCs. This work demonstrates that NPQ activation leads to effective photoprotection, promoting a photosystem II state characterized by a reduced probability of³Chl* formation, due to a decreased singlet excited state population, while maintaining an efficient quenching of the³Chl* eventually formed by carotenoids.► A measure of Chlorophyll and Carotenoid triplets upon NPQ induction is presented. ► All the PSII components undergo a decrease on triplet yield upon NPQ activation. ► The reduction on Chl triplet yield is reversible and correlated to qE. ► The structural rearrangements following NPQ do not lead to new triplet populations.

Keywords: Abbreviations; NPQ; non photochemical quenching; Car; carotenoid; Chl; chlorophyll; ZFS; zero field splitting; ISC; inter system crossing; ODMR; optically detected magnetic resonance; FDMR; fluorescence detected magnetic resonanceNPQ; Triplets; LHC; Carotenoid; FDMR; Physcomitrella patens

Adaptation of intracytoplasmic membranes to altered light intensity in Rhodobacter sphaeroides by Peter G. Adams; C. Neil Hunter (pp. 1616-1627).

The model photosynthetic bacterium Rhodobacter sphaeroides uses a network of bacteriochlorophyll (BChl)–protein complexes embedded in spherical intracytoplasmic membranes (ICM) to collect and utilise solar energy. We studied the effects of high- and low-light growth conditions, where BChl levels increased approximately four-fold from 1.6×10⁶ to 6.5×10⁶ molecules per cell. Most of this extra pigment is accommodated in the proliferating ICM system, which increases from approximately 274 to 1468 vesicles per cell. Thus, 16×10⁶nm² of specialised membrane surface area is made available for harvesting and utilising solar energy compared to 3×10⁶nm² under high-light conditions. Membrane mapping using atomic force microscopy revealed closely packed dimeric and monomeric reaction centre-light harvesting 1-PufX (RC-LH1-PufX) complexes in high-light ICM with room only for small clusters of LH2, whereas extensive LH2-only domains form during adaptation to low light, with the LH2/RC ratio increasing three-fold. The number of upper pigmented band (UPB) sites where membrane invagination is initiated hardly varied; 704 (5.8×10⁵ BChls/cell) and 829 (4.9×10⁵ BChls/cell) UPB sites per cell were estimated under high- and low-light conditions, respectively. Thus, the lower ICM content in high-light cells is a consequence of fewer ICM invaginations reaching maturity. Taking into account the relatively poor LH2-to-LH1 energy transfer in UPB membranes it is likely that high-light cells are relatively inefficient at energy trapping, but can grow well enough without the need to fully develop their photosynthetic membranes from the relatively inefficient UPB to highly efficient mature ICM.Display Omitted► We compared the effects of high and low light on Rba. sphaeroides membranes. ► Cellular pigment and membrane vesicles/cell increased more than 4-fold in low light. ► The number of sites of initiation of membrane growth is not affected by light. ► AFM shows closely packed core complexes in high-light membranes. ► Extensive LH2-only domains form following adaptation to low light.

Keywords: Abbreviations; AFM; atomic force microscopy; BChl(s); bacteriochlorophyll(s); B850; bacteriochlorophyll in light harvesting 2 complex with maximal absorption at 850; nm; B875; bacteriochlorophyll in light harvesting 1 complex with maximal absorption at 875; nm; EDTA; ethylenediamine tetraacetic acid; HEPES; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic Acid; ICM; intracytoplasmic membrane; LH1; light-harvesting 1 complex; LH2; light-harvesting 2 complex; Rba.; Rhodobacter; RC; reaction centre; RC-LH1; reaction centre-light-harvesting 1 complex; Rsp; .; Rhodospirillum; Rps.; Rhodopseudomonas; TEM; transmission electron microscopy; UPB; upper pigmented band; 2-D; two-dimensional; 3-D; three-dimensional; β-DDM; β-dodecylmaltoglucosideBacterial photosynthesis; Light harvesting; Light intensity; Membrane protein; Atomic force microscopy

Inhibition of complex I regulates the mitochondrial permeability transition through a phosphate-sensitive inhibitory site masked by cyclophilin D by Bo Li; Christiane Chauvin; Damien De Paulis; Frédéric De Oliveira; Abdallah Gharib; Guillaume Vial; Sandrine Lablanche; Xavier Leverve; Paolo Bernardi; Michel Ovize; Eric Fontaine (pp. 1628-1634).

Inhibition of the mitochondrial permeability transition pore (PTP) has proved to be an effective strategy for preventing oxidative stress-induced cell death, and the pore represents a viable cellular target for drugs. Here, we report that inhibition of complex I by rotenone is more effective at PTP inhibition than cyclosporin A in tissues that express low levels of the cyclosporin A mitochondrial target, cyclophilin D; and, conversely, that tissues in which rotenone does not affect the PTP are characterized by high levels of expression of cyclophilin D and sensitivity to cyclosporin A. Consistent with a regulatory role of complex I in the PTP-inhibiting effects of rotenone, the concentrations of the latter required for PTP inhibition precisely match those required to inhibit respiration; and a similar effect is seen with the antidiabetic drug metformin, which partially inhibits complex I. Remarkably (i) genetic ablation of cyclophilin D or its displacement with cyclosporin A restored PTP inhibition by rotenone in tissues that are otherwise resistant to its effects; and (ii) rotenone did not inhibit the PTP unless phosphate was present, in striking analogy with the phosphate requirement for the inhibitory effects of cyclosporin A [Basso et al. (2008) J. Biol. Chem. 283, 26307–26311]. These results indicate that inhibition of complex I by rotenone or metformin and displacement of cyclophilin D by cyclosporin A affect the PTP through a common mechanism; and that cells can modulate their PTP response to complex I inhibition by modifying the expression of cyclophilin D, a finding that has major implications for pore modulation in vivo.► Complex I inhibitors prevent mitochondrial permeability transition in most of tissues. ► Complex I inhibitors work best in the absence of Cyclophilin D. ► There is a synergy between Cyclosporin A and Complex I inhibitors. ► Cyclosporin A and Complex I inhibitors require Pi to prevent permeability transition .► Complex I might be a regulatory component of mitochondrial permeability transition.

Keywords: Abbreviations; ANT; adenine nucleotide translocator; COX; cytochrome oxidase; CRC; Ca; ²⁺; retention capacity; CsA; cyclosporin A; CypD; cyclophilin D; PT; permeability transition; PTP; permeability transition poreCyclophilin D; Cyclosporin A; Metformin; Mitochondria; Permeability transition; Rotenone

Photo-induced electron transfer from photosystem I to NADP⁺: Characterization and tentative simulation of the in vivo environment by Gwenaëlle Moal; Bernard Lagoutte (pp. 1635-1645).

The photoproduction of NADPH in photosynthetic organisms requires the successive or concomitant interaction of at least three proteins: photosystem I (PSI), ferredoxin (Fd) and ferredoxin:NADP⁺ oxidoreductase (FNR). These proteins and their surrounding medium have been carefully analysed in the cyanobacterium Synechocystis sp. PCC 6803. A high value of 550mg/ml was determined for the overall solute content of the cell soluble compartment. PSI and Fd are present at similar concentrations, around 500μM, whereas the FNR associated to phycobilisome is about 4 fold less concentrated. Membrane densities of FNR and trimeric PSI have been estimated to 2000 and 2550 per μm², respectively. An artificial confinement of Fd to PSI was designed using fused constructs between Fd and PsaE, a peripheral and stroma located PSI subunit. The best covalent system in terms of photocatalysed NADPH synthesis can be equivalent to the free system in a dilute medium. In a macrosolute crowded medium (375mg/ml), this optimized PSI/Fd covalent complex exhibited a huge superiority compared to the free system. This is a likely consequence of restrained diffusion constraints due to the vicinity of two out of the three protein partners. In vivo, Fd is the free partner, but the constant proximity between PSI and the phycobilisome associated FNR creates a similar situation, with two closely associated partners. This organization seems well adapted for an efficient in vivo production of the stable and fast diffusing NADPH.► General cell content of the cyanobacterium Synechocystis sp. PCC 6803 ► Quantification of the protein cascade catalysing the photo-reduction of NADP⁺ ► Designing ferredoxin photosystem I chimeras with a high electron transfer activity ► NADPH synthesis catalysed by chimeras versus free partners in a crowded medium

Keywords: Photosystem I; Ferredoxin; Ferredoxin–NADP-oxidoreductase; Gene fusion; Cell crowding

Thermodynamic constraints shape the structure of carbon fixation pathways by Arren Bar-Even; Avi Flamholz; Elad Noor; Ron Milo (pp. 1646-1659).

Thermodynamics impose a major constraint on the structure of metabolic pathways. Here, we use carbon fixation pathways to demonstrate how thermodynamics shape the structure of pathways and determine the cellular resources they consume. We analyze the energetic profile of prototypical reactions and show that each reaction type displays a characteristic change in Gibbs energy. Specifically, although carbon fixation pathways display a considerable structural variability, they are all energetically constrained by two types of reactions: carboxylation and carboxyl reduction. In fact, all adenosine triphosphate (ATP) molecules consumed by carbon fixation pathways – with a single exception – are used, directly or indirectly, to power one of these unfavorable reactions. When an indirect coupling is employed, the energy released by ATP hydrolysis is used to establish another chemical bond with high energy of hydrolysis, e.g. a thioester. This bond is cleaved by a downstream enzyme to energize an unfavorable reaction. Notably, many pathways exhibit reduced ATP requirement as they couple unfavorable carboxylation or carboxyl reduction reactions to exergonic reactions other than ATP hydrolysis. In the most extreme example, the reductive acetyl coenzyme A (acetyl-CoA) pathway bypasses almost all ATP-consuming reactions. On the other hand, the reductive pentose phosphate pathway appears to be the least ATP-efficient because it is the only carbon fixation pathway that invests ATP in metabolic aims other than carboxylation and carboxyl reduction. Altogether, our analysis indicates that basic thermodynamic considerations accurately predict the resource investment required to support a metabolic pathway and further identifies biochemical mechanisms that can decrease this requirement.► Different reaction types display a characteristic change in Gibbs energy. ► Carbon fixation is constrained by carboxylations and carboxyl reductions. ► All ATP consumption reactions are coupled to one of these unfavorable reactions. ► Coupling unfavorable reactions to exergonic reactions other than ATP hydrolysis.

Keywords: Abbreviations; Δ; _r; G′; ^m; Transformed Gibbs energy of a reaction under reactant concentrations of 1; mM.Reduction potential; Carbon fixation; Thermodynamic favorability; Exergonism; Reaction coupling; ATP cost

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak by Martin Jastroch; Verena Hirschberg; Martin Klingenspor (pp. 1660-1670).

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 — HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.► Characterization of a novel mammalian cell system to test the function of UCP1. ► Mouse UCP1 does not contribute to mitochondrial basal proton leak. ► Quantification and meta-analysis of UCP1 catalytic centre activities ► Mathematical approach to the relation between proton leak and membrane potential

Keywords: UCP1; Human embryonic kidney cell; Adenine nucleotide translocase (ANT); Proton leak kinetics; Basal proton leak

Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations by Sergey Vassiliev; Tatiana Zaraiskaya; Doug Bruce (pp. 1671-1678).

The Mn₄Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn₄Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn₄Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca2⁺ and Cl⁻ from the active site.► First SMD simulations of solvated photosystem II. ► Determined the energetic barriers for water permeation though PSII. ► Identified the main constriction sites in water pathways. ► None of the channels permit unrestricted access of water to the OEC.

Keywords: Abbreviations; PSII; photosystem II; PMF; potential of mean force; SMD; steered molecular dynamics; OEC; oxygen-evolving complexPhotosystem II; Water permeation; Molecular dynamics

l-Lactate metabolism in HEP G2 cell mitochondria due to thel-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria by Roberto Pizzuto; Gianluca Paventi; Carola Porcile; Daniela Sarnataro; Aurora Daniele; Salvatore Passarella (pp. 1679-1690).

As part of an ongoing study ofl-lactate metabolism both in normal and in cancer cells, we investigated whether and howl-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess anl-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondriall-LDHs were found to differ from one another in their saturation kinetics. Having shown thatl-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammoniuml-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediatedl-lactate transport in these mitochondria. Occurrence of thel-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising froml-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role forl-LAC in Hep G2-M.► The existence of the mitochondriall-LDH (ml-LDH) in Hep G2 cells is shown. ► ml-LDH metabolizesl-lactate that in distinction with pyruvate enters mitochondria. ► Thel-lactate/pyruvate shuttle is reconstructed by which NADH is oxidized. ► Oxaloacetate derived from taken upl-lactate is exported outside mitochondria. ► Mitochondriall-lactate transport and metabolism play an anaplerotic role.

Keywords: l; -LDH; Mitochondrion; l; -Lactate/pyruvate shuttle; Hep G2 cell; Cancer

Mitochondrial DNA sequence variation is associated with free-living activity energy expenditure in the elderly by Gregory J. Tranah; Ernest T. Lam; Shana M. Katzman; Michael A. Nalls; Yiqiang Zhao; Daniel S. Evans; Jennifer S. Yokoyama; Ludmila Pawlikowska; Pui-Yan Kwok; Sean Mooney; Stephen Kritchevsky; Bret H. Goodpaster; Anne B. Newman; Tamara B. Harris; Todd M. Manini; Steven R. Cummings (pp. 1691-1700).

The decline in activity energy expenditure underlies a range of age-associated pathological conditions, neuromuscular and neurological impairments, disability, and mortality. The majority (90%) of the energy needs of the human body are met by mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS is dependent on the coordinated expression and interaction of genes encoded in the nuclear and mitochondrial genomes. We examined the role of mitochondrial genomic variation in free-living activity energy expenditure (AEE) and physical activity levels (PAL) by sequencing the entire (~16.5 kilobases) mtDNA from 138 Health, Aging, and Body Composition Study participants. Among the common mtDNA variants, the hypervariable region 2 m.185G>A variant was significantly associated with AEE (p=0.001) and PAL (p=0.0005) after adjustment for multiple comparisons. Several unique nonsynonymous variants were identified in the extremes of AEE with some occurring at highly conserved sites predicted to affect protein structure and function. Of interest is the p.T194M, CytB substitution in the lower extreme of AEE occurring at a residue in the Qi site of complex III. Among participants with low activity levels, the burden of singleton variants was 30% higher across the entire mtDNA and OXPHOS complex I when compared to those having moderate to high activity levels. A significant pooled variant association across the hypervariable 2 region was observed for AEE and PAL. These results suggest that mtDNA variation is associated with free-living AEE in older persons and may generate new hypotheses by which specific mtDNA complexes, genes, and variants may contribute to the maintenance of activity levels in late life.► Human mitochondrial sequence variation is associated with energy expenditure. ► Highly conserved substitutions are found in the extremes of energy expenditure. ► Aggregate complex I and HV2 sequences are associated with energy expenditure. ► Total mtDNA and complex I singleton burden was elevated in sedentary subjects.

Keywords: Metabolic rate; Energy expenditure; Mitochondria; mtDNA; Oxidative phosphorylation; DNA sequencing

Heterologously expressed arsenite oxidase: A system to study biogenesis and structure/function relationships of the enzyme family by Robert van Lis; Wolfgang Nitschke; Thomas P. Warelow; Line Capowiez; Joanne M. Santini; Barbara Schoepp-Cothenet (pp. 1701-1708).

Studies of native arsenite oxidases from Ralstonia sp. S22 and Rhizobium sp. NT-26 raised two major questions. The first one concerns the mode of the enzyme's membrane-association. It has been suggested that a hypothetical not conserved protein could account for this variable association. Expression of the wild type arsenite oxidase in Escherichia coli allowed us to study the cellular localization of this enzyme in the absence of such a hypothetical partner. The results with the Ralstonia sp. S22 enzyme suggest that no additional protein is required for membrane association. The second question addresses the influence of the disulfide bridge in the small Rieske subunit, conspicuously absent in the Rhizobium sp. NT-26 enzyme, on the properties of the [2Fe–2S] center. The disulfide bridge is considered to be formed only after translocation of the enzyme to the periplasm. To address this question we thus first expressed the enzyme in the absence of its Twin-arginine translocation signal sequence. The spectral and redox properties of the cytoplasmic enzyme are unchanged compared to the periplasmic one. We finally studied a disulfide bridge mutant, Cys106Ala, devoid of the first Cys involved in the disulfide bridge formation. This mutation, proposed to have a strong effect on redox and catalytic properties of the Rieske protein in Rieske/cyt b complexes, had no significant effect on properties of the Rieske protein from arsenite oxidase. Our present results demonstrate that the effects attributed to the disulfide bridge in the Rieske/cyt b complexes are likely to be secondary effects due to conformational changes.► We expressed the arsenite oxidase from Ralstonia sp. S22 in Escherichia coli. ► Results on wild type and mutated Aio pertaining to the biogenesis of the enzyme were obtained. ► We analyzed Aio's structure/function relationships by comparing wild type and mutant enzymes. ► We propose that the uncleaved Tat sequence of AioB anchors the enzyme to the membrane. ► The S–S bridge in the Rieske protein affects neither redox nor structural/functional properties.

Keywords: Abbreviations; As; ^III; Arsenite; AioAB; arsenite oxidase; SDS-PAGE; Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis; FPLC; Fast protein liquid chromatography; PCR; Polymerase Chain Reaction; Tat; Twin-arginine translocationArsenite oxidase; Rieske protein; Disulfide bridge; EPR; Tat signal leader sequence

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BBA - Bioenergetics (v.1817, #9)