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BBA - Bioenergetics (v.1777, #10)

Editorial Board (pp. ii).

Coupling of electron transfer to proton uptake at the Q_B site of the bacterial reaction center: A perspective from FTIR difference spectroscopy by Eliane Nabedryk; Jacques Breton (pp. 1229-1248).

FTIR difference spectroscopy provides a unique approach to study directly protonation/deprotonation events of carboxylic acids involved in the photochemical cycle of membrane proteins, such as the bacterial photosynthetic reaction center (RC). In this work, we review the data obtained by light-induced FTIR difference spectroscopy on the first electron transfer to the secondary quinone Q_B in native RCs and a series of mutant RCs. We first examine the approach of isotope-edited FTIR spectroscopy to investigate the binding site of Q_B. This method provides highly specific IR vibrational fingerprints of the bonding interactions of the carbonyls of Q_B and Q_B⁻ with the protein. The same isotope-edited IR fingerprints for the carbonyls of neutral Q_B have been observed for native Rhodobacter sphaeroides RCs and several mutant RCs at the Pro-L209, Ala-M260, or Glu-L212/Asp-L213 sites, for which X-ray crystallography has found the quinone in the proximal position. It is concluded that at room temperature Q_B occupies a single binding site that fits well the description of the proximal site derived from X-ray crystallography and that the conformational gate limiting the rate of the first electron transfer from Q_A⁻Q_B to Q_AQ_B⁻ cannot be the movement of Q_B from its distal to proximal site. Possible alternative gating mechanisms are discussed. In a second part, we review the contribution of the various experimental measurements, theoretical calculations, and molecular dynamics simulations which have been actively conducted to propose which amino acid side chains near Q_B could be proton donors/acceptors. Further, we show how FTIR spectroscopy of mutant RCs has directly allowed several carboxylic acids involved in proton uptake upon first electron transfer to Q_B to be identified. Owing to the importance of a number of residues for high efficiency of coupled electron transfer reactions, the photoreduction of Q_B was studied in a series of single mutant RCs at Asp-L213, Asp-L210, Asp-M17, Glu-L212, Glu-H173, as well as combinations of these mutations in double and triple mutant RCs. The same protonation pattern was observed in the 1760–1700 cm−¹ region of the Q_B⁻/Q_B spectra of native and several mutant (DN-L213, DN-L210, DN-M17, EQ-H173) RCs. However, it was drastically modified in spectra of mutants lacking Glu at L212. The main conclusion of this work is that in native RCs from Rb. sphaeroides, Glu-L212 is the only carboxylic acid residue that contributes to proton uptake at all pH values (from pH 4 to pH 11) in response to the formation of Q_B⁻. Another important result is that the residues Asp-L213, Asp-L210, Asp-M17, and Glu-H173 are mostly ionized in the Q_B state at neutral pH and do not significantly change their protonation state upon Q_B⁻ formation. In contrast, interchanging Asp and Glu at L212 and L213 (i.e., in the so-called swap mutant) led to the identification of a novel protonation pattern of carboxylic acids: at least four individual carboxylic acids were affected by Q_B reduction. The pH dependence of IR carboxylic signals in the swap mutant demonstrates that protonation of Glu-L213 occurred at pH >5 whereas that of Asp-L212 occurred over the entire pH range from 8 to 4. In native RCs from Rhodobacter sphaeroides, a broad positive IR continuum around 2600 cm−¹ in the Q_B⁻/Q_B steady-state FTIR spectrum in¹H₂O was assigned to delocalized proton(s) in a highly polarizable hydrogen-bonded network. The possible relation of the IR continuum band to the carboxylic acid residues and to bound water molecules involved in the proton transfer pathway was investigated by testing the robustness of this band to different mutations of acids. The presence of the band is not correlated with the localization of the proton on Glu-L212. The largest changes of the IR continuum were observed in single and double mutant RCs where Asp-L213 is not present. It is proposed that the changes observed in the mutant RCs with respect to native RCs reflect the specific role of bound protonated water molecule(s) located in the vicinity of Asp-L213 and undergoing hydrogen-bond changes in the network.

Keywords: Abbreviations; P; primary electron donor; BChl; bacteriochlorophyll; Bphe; bacteriopheophytin; H; _A; intermediate electron acceptor; Q; _A; (Q; _B; ), primary, (secondary), quinone electron acceptor; Q; n; , ubiquinone-; n; :; 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone; RC; reaction center; Rb; .; Rhodobacter; B; .; Blastochloris; ET; electron transfer; FTIR; Fourier transform infrared; RS; rapid-scan; TR; time-resolved; EPR; electron paramagnetic resonance; ENDOR; electron nuclear double resonanceFTIR; Reaction center; Quinone; Site-directed mutant; Carboxylic acid; Protonation event

Membrane phospholipids, lipoxidative damage and molecular integrity: A causal role in aging and longevity by Reinald Pamplona (pp. 1249-1262).

Nonenzymatic molecular modifications induced by reactive carbonyl species (RCS) generated by peroxidation of membrane phospholipids acyl chains play a causal role in the aging process. Most of the biological effects of RCS, mainly α,β-unsaturated aldehydes, di-aldehydes, and keto-aldehydes, are due to their capacity to react with cellular constituents, forming advanced lipoxidation end-products (ALEs). Compared to reactive oxygen and nitrogen species, lipid-derived RCS are stable and can diffuse within or even escape from the cell and attack targets far from the site of formation. Therefore, these soluble reactive intermediates, precursors of ALEs, are not only cytotoxic per se, but they also behave as mediators and propagators of oxidative stress and cellular and tissue damage. The consequent loss-of-function and structural integrity of modified biomolecules can have a wide range of downstream functional consequences and may be the cause of subsequent cellular dysfunctions and tissue damage. The causal role of ALEs in aging and longevity is inferred from the findings that follow: a) its accumulation with aging in several tissues and species; b) physiological interventions (dietary restriction) that increase longevity, decrease ALEs content; c) the longer the longevity of a species, the lower is the lipoxidation-derived molecular damage; and finally d) exacerbated levels of ALEs are associated with pathological states.

Keywords: Abbreviations; AA; arachidonic acid; AKR; aldo-keto reductase; ALDH; aldehyde dehydrogenase; ALEs; advanced lipoxidation end-product; ARE; antioxidant response element; CBP; CREB-binding protein; CR; caloric restriction; CREB; cAMP-responsive element-binding factor; DBI; double bond index; 2-DE-WB-MALDI-TOF; two dimensional electrophoresis-western blot-matrix assisted laser desorption ionization time of flight; DHA; docosahexaenoic acid; EGFR; epidermal growth factor receptor; EPA; eicosapentaenoic acid; ERK; extracellular signal-regulated kinase; GFAP; glial fibrillary acidic protein; GPX; glutathione peroxidase; GSH; glutathione; GST; glutathione-; S; -transferase; 2-HH; 2-hydroxyheptanal; 4-HHE; 4-hydroxyhexenal; 4-HNE; 4-hydroxy-; trans; -2-nonenal; HSFs; heat-shock transcriptional factors; HSPs; heat-shock proteins; JNK; c-Jun N-terminal kinase; Keap1; Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1; LA; linoleic acid; MAPK; mitogen-activated protein kinase; MDA; malondialdehyde; M1dG; malondialdehyde-deoxyguanosine; NF-kB; nuclear factor-kB; Nrf2; nuclear factor-erythroid 2 (NF-E2)-related factor 2; ONE; 4-oxo-; trans; -2-nonenal; PDGFR; platelet-derived growth factor receptor; PHGPX; phospholipids hydroperoxide glutathione peroxidase; PI; peroxidizability index; PUFA; polyunsaturated fatty acid; RCS; reactive carbonyl species; RONS; reactive nitrogen species; ROS; reactive oxygen species; SAM; senescent accelerated mouse; SOD; superoxide dismutase; SREBP; sterol regulatory element-binding protein; TGFbeta-1; tissue growth factor beta1; TKR; tyrosine kinase receptors; UCPs; uncoupling proteinsAdvanced lipoxidation end-product; Aging; Double bond index; Free radical; Lipid oxidation; Longevity; Membrane unsaturation; Mitochondria; Molecular damage; Lipoxidation; Peroxidizability index; Reactive carbonyl species; Unsaturated aldehyde

Photoprotection in higher plants: The putative quenching site is conserved in all outer light-harvesting complexes of Photosystem II by Milena Mozzo; Francesca Passarini; Roberto Bassi; Herbert van Amerongen; Roberta Croce (pp. 1263-1267).

In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575–578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.

Keywords: Abbreviations; Chl; chlorophyll; Lhc; light-harvesting complexPhotosynthesis; Photosystem II; Light-harvesting complex; Photoprotection

Nitrite–nitric oxide control of mitochondrial respiration at the frontier of anoxia by Abdelilah Benamar; Hardy Rolletschek; Ljudmilla Borisjuk; Marie-Hélène Avelange-Macherel; Gilles Curien; H. Ahmed Mostefai; Ramaroson Andriantsitohaina; David Macherel (pp. 1268-1275).

Actively respiring animal and plant tissues experience hypoxia because of mitochondrial O₂ consumption. Controlling oxygen balance is a critical issue that involves in mammals hypoxia-inducible factor (HIF) mediated transcriptional regulation, cytochrome oxidase (COX) subunit adjustment and nitric oxide (NO) as a mediator in vasodilatation and oxygen homeostasis. In plants, NO, mainly derived from nitrite, is also an important signalling molecule. We describe here a mechanism by which mitochondrial respiration is adjusted to prevent a tissue to reach anoxia. During pea seed germination, the internal atmosphere was strongly hypoxic due to very active mitochondrial respiration. There was no sign of fermentation, suggesting a down-regulation of O₂ consumption near anoxia. Mitochondria were found to finely regulate their surrounding O₂ level through a nitrite-dependent NO production, which was ascertained using electron paramagnetic resonance (EPR) spin trapping of NO within membranes. At low O₂, nitrite is reduced into NO, likely at complex III, and in turn reversibly inhibits COX, provoking a rise to a higher steady state level of oxygen. Since NO can be re-oxidized into nitrite chemically or by COX, a nitrite–NO pool is maintained, preventing mitochondrial anoxia. Such an evolutionarily conserved mechanism should have an important role for oxygen homeostasis in tissues undergoing hypoxia.

Keywords: Abbreviations; CPTIO; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide; COX; cytochrome oxidase; EPR; electron paramagnetic resonance; NO; nitric oxideMitochondria; Respiration; Hypoxia; Anoxia; Nitric oxide; Nitrite

Impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs onmitochondrial energy metabolism by Sven Wolfgang Sauer; Juergen G. Okun; Georg F. Hoffmann; Stefan Koelker; Marina A. Morath (pp. 1276-1282).

Accumulation of organic acids as well as their CoA and carnitine esters in tissues and body fluids is a common finding in organic acidurias, beta-oxidation defects, Reye syndrome, and Jamaican vomiting sickness. Pathomechanistic approaches for these disorders have been often focused on the effect of accumulating organic acids on mitochondrial energy metabolism, whereas little is known about the pathophysiologic role of short- and medium-chain acyl-CoAs and acylcarnitines. Therefore, we investigated the impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs on central components of mitochondrial energy metabolism, namely alpha-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and single enzyme complexes I-V of respiratory chain. Although at varying degree, all acyl-CoAs had an inhibitory effect on pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity. Effect sizes were critically dependent on chain length and number of functional groups. Unexpectedly, octanoyl-CoA was shown to inhibit complex III. The inhibition was noncompetitive regarding reduced ubiquinone and uncompetitive regarding cytochrome c. In addition, octanoyl-CoA caused a blue shift in the gamma band of the absorption spectrum of reduced complex III. This effect may play a role in the pathogenesis of medium-chain and multiple acyl-CoA dehydrogenase deficiency, Reye syndrome, and Jamaican vomiting sickness which are inherited and acquired conditions of intracellular accumulation of octanoyl-CoA.

Keywords: Abbreviations; CoA; coenzyme A; KGDHc; alpha; -ketoglutarate dehydrogenase complex; PDHc; pyruvate dehydrogenase complex; DBH; decylubihydroquinone; TCA; tricarboxylic acid; SMOAA; short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs; GCDH; glutaryl-CoA dehydrogenase; MMA; methylmalonic aciduria; A-CoA; Acetyl-CoA; P-CoA; propionyl-CoA; B-CoA; butyryl-CoA; I-CoA; isovaleryl-CoA; H-CoA; hexanoyl-CoA; O-CoA; octanoyl-CoA; M-CoA; malonyl-CoA; MM-CoA; methylmalonyl-CoA; G-CoA; glutaryl-CoA; SMPs; submitochondrial particles from bovine heart; AIC; Akaike's Information Criterion; MCAD; medium-chain acyl-CoA dehydrogenase; ETF; electron transfer flavoproteinOrganic acid; Acylcarnitine; Acyl-CoAs; Octanoyl-CoA; Mitochondrial energy metabolism; Respiratory chain; Complex III; Alpha; -ketoglutarate dehydrogenase complex; Pyruvate dehydrogenase complex

Kinetic activation of yeast mitochondriald-lactate dehydrogenase by carboxylic acids by Arnaud Mourier; Julie Vallortigara; Edgar D. Yoboue; Michel Rigoulet; Anne Devin (pp. 1283-1288).

Aerobically grown yeast cells express mitochondrial lactate dehydrogenases that localize to the mitochondrial inner membrane. Thed-lactate dehydrogenase is a zinc-flavoprotein with high acceptor specificity for cytochrome c, that catalyzes the oxidation ofd-lactate into pyruvate. In this paper, we show that mitochondrial respiratory rate in phosphorylating or non-phosphorylating conditions withd-lactate as substrate is stimulated by carboxylic acids. This stimulation does not affect the yield of oxidative phosphorylation. Furthermore, this stimulation lies at the level of thed-lactate dehydrogenase. It is non-competitive, hyperbolic and its dimension is directly related to the number of carboxylic groups on the activator. The physiological meaning of such a regulation is discussed.

Keywords: d; -lactate dehydrogenase; Mitochondria; Yeast; Kinetic activation; Carboxyls groups

A peptide containing residues 26–44 of tau protein impairs mitochondrial oxidative phosphorylation acting at the level of the adenine nucleotide translocator by A. Atlante; G. Amadoro; A. Bobba; L. de Bari; V. Corsetti; G. Pappalardo; E. Marra; P. Calissano; S. Passarella (pp. 1289-1300).

Having confirmed that adenovirus-mediated overexpression of NH₂-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH₂-derived tau peptides, i.e. NH₂-26–44 and NH₂-1–25 fragments, affect mitochondrial function. We tested both fragments on each step of the processes leading to ATP synthesis via oxidative phosphorylation: i) electron flow via the respiratory chain from physiological substrates to oxygen with the activity of each individual complex of the respiratory chain investigated in some detail, ii) membrane potential generation arising from externally added succinate and iii) the activity of both the adenine nucleotide translocator and iv) ATP synthase. Oxidative phosphorylation is not affected by NH₂-1–25 tau fragment, but dramatically impaired by NH₂-26–44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH₂-26–44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH₂-26–44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria.

Keywords: Abbreviations; AA; antimycin A; AD; Alzheimer Disease; ANT; adenine nucleotide translocator; Ap5A; P; ₁; ,P; ₅; -Di(adenosine-5′)penta-phosphate; ATP D.S.; ATP detecting system; ATR; atractyloside; BME; basal medium Eagle; β; -OH,; β-hydroxybutyrate; CGC; cerebellar granule cells; CN; ⁻; potassium cyanide; Control; trace in the absence of NH; ₂; -tau fragments; COX; cytochrome oxidase; MERS; mersalyl; NMDA; N; -methyl-; d; -aspartate; OLIGO; oligomycin; PBS; phosphate buffer saline medium; RAM; rotenone plus antimycin A plus mixothiazole; RCI; respiratory control index; ROT; rotenone; S.D.; standard deviation; SUCC; succinateMitochondria; Tau fragment; Cerebellar granule cells; Oxidative phosphorylation; ATP synthesis; Neurotoxicity

Δψ and ΔpH are equivalent driving forces for proton transport through isolated F₀ complexes of ATP synthases by Alexander Wiedenmann; Peter Dimroth; Christoph von Ballmoos (pp. 1301-1310).

The membrane-embedded F₀ part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F₀ complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F₀ part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F₁F₀ holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H⁺-transport through F₀. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H⁺/(s×F₀) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H⁺-transport at initial rates of 6300 H⁺/(s×F₀) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.

Keywords: Abbreviations; DCCD; dicyclohexylcarbodiimide; CCCP; carbonyl cyanide; m; -chlorophenylhydrazone; TBT; tributlytinATP synthase; F; ₀; part; H; ⁺; -transport; Pyranine; Driving force

The terminal reaction cascade of water oxidation: Proton and oxygen release by Juergen Clausen; Wolfgang Junge (pp. 1311-1318).

In cyanobacteria, algae and plants Photosystem II produces the oxygen we breathe. Driven and clocked by light quanta, the catalytic Mn₄Ca-tyrosine centre accumulates four oxidising equivalents before it abstracts four electrons from water, liberating dioxygen and protons. Aiming at intermediates of the terminal four-electron cascade, we previously have suppressed this reaction by elevating the oxygen pressure, thereby stabilising one redox intermediate. Here, we established a similar suppression by increasing the proton concentration. Data were analysed in terms of only one (peroxy) redox intermediate between the fourfold oxidised Mn₄Ca-tyrosine centre and oxygen release. The surprising result was that the release into the bulk of one proton per dioxygen is linked to the first and rate-limiting electron transfer in the cascade rather than to the second which produces free oxygen. The penultimate intermediate might thus be conceived as a fully deprotonated peroxy-moiety.

Keywords: Photosynthesis; Water oxidation; Proton release; Photosystem II; Intermediate; Oxygen

Direct catalytic electrochemistry of sulfite dehydrogenase: Mechanistic insights and contrasts with related Mo enzymes by Trevor D. Rapson; Ulrike Kappler; Paul V. Bernhardt (pp. 1319-1325).

Under hydrodynamic electrochemical conditions with slow cyclic voltammetry sweep rates we have been able to probe catalytic events at the molybdenum active site of sulfite dehydrogenase (SDH) from Starkeya novella adsorbed on an edge plane graphite electrode within a polylysine film. The electrochemically driven catalytic behaviour of SDH mirrors that seen in solution assays suggesting that the adsorbed enzyme retains its native activity. However, at high sulfite concentrations, the voltammetric waveform transforms from the expected sigmoidal profile to a peak-shaped response, similar to that reported for the molybdenum enzymes DMSO reductase and nitrate reductase (NarGHI and NapAB) where a redox reaction at the active site has been associated with a switch to lower activity at high overpotentials. This is the first time a similar phenomenon has been observed in a Mo-containing oxidase/dehydrogenase, which raises a number of interesting mechanistic problems. The potential at which the activity of SDH becomes attenuated only emerges at saturating substrate conditions and occurs at a potential ( ca. + 320mV vs NHE) well removed from any known redox couple in the enzyme. These results cannot be explained by the same mechanism adopted for DMSO reductase and nitrate reductase catalysis.

Keywords: Abbreviations; SDH; sulfite dehydrogenase; DMSO; dimethyl sulfoxide; Cys; cysteine; HSO; human sulfite oxidase; CSO; chicken sulfite oxidase; PSO; plant sulfite oxidase; EDTA; ethylenediamine-tetraacetete; tris; tris(hydroxymethyl)-methylammonium; NHE; normal hydrogen electrode; RDV; rotating disk voltammetry; EPG; edge plane pyrolytic graphite; DDAB; didodecyldimethylammonium bromide; SDH; sulfite dehydrogenase; DMSO; dimethyl sulfoxide; Cys; cysteine; HSO; human sulfite oxidase; CSO; chicken sulfite oxidase; PSO; plant sulfite oxidase; EDTA; ethylenediamine-tetraacetete; tris; tris(hydroxymethyl)-methylammonium; NHE; normal hydrogen electrode; RDV; rotating disk voltammetry; EPG; edge plane pyrolytic graphite; DDAB; didodecyldimethylammonium bromide; Tyr; tyrosine; Phe; phenylalanineMolybdenum; Enzyme; Electrochemistry

Structural and functional characterization of F_oF₁-ATP synthase on the extracellular surface of rat hepatocytes by Roberto Mangiullo; Antonio Gnoni; Antonella Leone; Gabriele V. Gnoni; Sergio Papa; Franco Zanotti (pp. 1326-1335).

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F_oF₁-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F₁ (α/β and γ) and F_o (F_oI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF₁ is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H⁺ and are inhibited by F₁ and F_o-targeting inhibitors. We hypothesise that ecto-F_oF₁-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.

Keywords: ATP synthesis and hydrolysis; Ectopic F; _o; F; ₁; -ATP synthase; H+ conduction; Plasma membrane; Rat hepatocyte

Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans by Freya A. Bundschuh; Klaus Hoffmeier; Bernd Ludwig (pp. 1336-1343).

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba₃-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa₃-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa₃-type cytochrome c oxidase and the ba₃-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.

Keywords: Abbreviations; COX; cytochrome; c; oxidase; QOX; quinol oxidaseRespiratory chain; Heme/copper oxidase; Oxidase biogenesis; Heme; a; Binuclear centre; Heme incorporation

Occurrence and function of the orange carotenoid protein in photoprotective mechanisms in various cyanobacteria by Clémence Boulay; Leyla Abasova; Christophe Six; Imre Vass; Diana Kirilovsky (pp. 1344-1354).

Excess light is harmful for photosynthetic organisms. The cyanobacterium Synechocystis PCC 6803 protects itself by dissipating the excess of energy absorbed by the phycobilisome, the water-soluble antenna of Photosystem II, into heat decreasing the excess energy arriving to the reaction centers. Energy dissipation results in a detectable decrease of fluorescence. The soluble Orange Carotenoid Protein (OCP) is essential for this blue-green light induced mechanism. OCP genes appear to be highly conserved among phycobilisome-containing cyanobacteria with few exceptions. Here, we show that only the strains containing a whole OCP gene can perform a blue-light induced photoprotective mechanism under both iron-replete and iron-starvation conditions. In contrast, strains containing only N-terminal and/or C-terminal OCP-like genes, or no OCP-like genes at all lack this light induced photoprotective mechanism and they were more sensitive to high-light illumination. These strains must adopt a different strategy to longer survive under stress conditions. Under iron starvation, the relative decrease of phycobiliproteins was larger in these strains than in the OCP-containing strains, avoiding the appearance of a population of dangerous, functionally disconnected phycobilisomes. The OCP-containing strains protect themselves from high light, notably under conditions inducing the appearance of disconnected phycobilisomes, using the energy dissipation OCP-phycobilisome mechanism.

Keywords: Cyanobacteria; Iron starvation; Non-photochemical quenching; Orange-carotenoid-protein; Photoprotection; Photosystem II

Spectroscopic properties of the peridinins involved in chlorophyll triplet quenching in high-salt peridinin–chlorophyll a-protein from Amphidinium carterae as revealed by optically detected magnetic resonance, pulse EPR and pulse ENDOR spectroscopies by Marilena Di Valentin; Stefano Ceola; Enrico Salvadori; Giancarlo Agostini; Giorgio Mario Giacometti; Donatella Carbonera (pp. 1355-1363).

The photoexcited triplet state of the carotenoid peridinin in the high-salt peridinin–chlorophyll a-protein (HSPCP) of the dinoflagellate Amphidinium carterae was investigated by ODMR (optically detected magnetic resonance), pulse EPR and pulse ENDOR spectroscopies. The properties of peridinins associated to the triplet state formation in HSPCP were compared to those of peridinins involved in triplet state population in the main-form peridinin–chlorophyll protein (MFPCP), previously reported. In HSPCP no signals due to the presence of chlorophyll triplet state have been detected, during either steady-state illumination or laser-pulse excitation, meaning that peridinins play the photo-protective role with 100% efficiency as in MFPCP. The general spectroscopic features of the peridinin triplet state are very similar in the two complexes and allow drawing the conclusion that the triplet formation pathway and the triplet localization in one specific peridinin in each subcluster are the same in HSPCP and MFPCP. However some significant differences also emerged from the analysis of the spectra. Zero field splitting parameters of the peridinin triplet states are slightly smaller in HSPCP and small changes are also observed for the hyperfine splittings measured by pulse ENDOR and assigned to the β-protons belonging to one of the two methyl groups present in the conjugated chain, ( a_iso=10.3 MHz in HSPCP vs a_iso=10.6 MHz in MFPCP). The differences are explained in terms of local distortion of the tails of the conjugated chains of the peridinin molecules, in agreement with the conformational data resulting from the X-ray structures of the two complexes.

Keywords: Abbreviations; PID, peridinin; Chl, chlorophyll; PCP, peridinin–chlorophyll protein; A. carterae; ,; Amphidinium carterae; MFPCP, main-form PCP; HSPCP, high-salt PCP; ODMR, optically detected magnetic resonance; FDMR, fluorescence detected magnetic resonance; ADMR, absorption detected magnetic resonance; ZFS, zero field splitting; ISC, intersystem crossing; ESE, electron spin echo; ENDOR, electron nuclear double resonance; TR-EPR, Time-resolved Electron Paramagnetic Resonance; hf: hyperfine; hfcs: hyperfine constantsPeridinin; Carotenoid; Triplet; ODMR; EPR; ENDOR; HSPCP; MFPCP

Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase by Ramirez Pablo Ramírez; Nicolas Mano; Rafael Andreu; Tautgirdas Ruzgas; Adam Heller; Lo Gorton; Sergey Shleev (pp. 1364-1369).

Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modified gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied under anaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry. On cyclic voltammograms nonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observed corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively. The immobilized BOD was differently oriented on the two electrodes and its catalysis of O₂-electroreduction was also massively different. On SPGE, where most of the enzyme was oriented with the T1 copper site proximal to the carbon with a quite slow ET process, well-pronounced DET-bioelectroreduction of O₂ was observed, starting already at >700 mV vs. NHE. In contrast, on MPA-gold most of the enzyme was oriented with its T2/T3 copper cluster proximal to the metal. Indeed, there was little DET-based catalysis of O₂-electroreduction, even though the ET between the MPA-gold and the T2/T3 copper cluster of BOD was similar to that observed for the T1 site at SPGE. When BOD actively catalyzes the O₂-electroreduction, the redox potential of its T1 site is 690 mV vs. NHE and that of one of its T2/T3 copper centers is 390 mV vs. NHE. The redox potential of the T2/T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs. NHE. These values, combined with the observed biocatalytic behavior, strongly suggest an uphill intra-molecular electron transfer from the T1 site to the T2/T3 cluster during the catalytic turnover of the enzyme.

Keywords: Abbreviations; BOD; bilirubin oxidase; NI; native intermediate; PI; peroxy intermediate; FR; fully reduced BOD; RF; resting form; DET; direct electron transfer; ET; electron transfer; IET; intra-molecular electron transfer; CV; cyclic voltammogram; E; _m; midpoint redox potential; E; _T1; ,; E; _T2; ,; E; _T3; , and; E; _T2/T3; redox potentials of the T1, T2, T3 sites and the T2/T3 cluster, respectively; Δ; E; _p; peak separation between anodic and cathodic peaks; k; ₀; standard electron transfer rate constant; k; _DET; heterogeneous DET rate constant; Γ; surface concentration; j; _cat; biocatalytic current density; j; cat; max; maximum biocatalytic current density; Δ; j; cat(; +; F−; ₎; differences in biocatalytic current densities in the absence and presence of F; ⁻; EDC; N; -(3-dimethylaminopropyl)-; N; ′-ethylcarbodiimide hydrochloride; NHS; N; -hydroxysuccinimide; AMTP; 4-aminothiophenol; MHOL; 6-mercapto-1-hexanol; MPA; 3-mercaptopropionic acid; DT; 1-decanethiol; SPGE; spectrographic graphite electrode; MPA-gold; 3-mercaptopropionic acid-modified gold electrodeBilirubin oxidase; Bioelectrocatalysis; O; ₂; -electroreduction; Electron transfer kinetics

Defective assembly of a hybrid vacuolar H⁺-ATPase containing the mouse testis-specific E1 isoform and yeast subunits by Kazuhiro Hayashi; Ge-Hong Sun-Wada; Yoh Wada; Mayumi Nakanishi-Matsui; Masamitsu Futai (pp. 1370-1377).

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V_o and catalytic V₁ sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098–18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the V_o subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V₁ sector was still associated with V_o at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V₁ from V_o in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.

Keywords: Abbreviations; V-ATPase; vacuolar-type proton pumping ATPase; MES; 4-morpholineethanesulfonic acid; E; 1; testis-specific V-ATPase subunit; E; isoform; E; 2; ubiquitous V-ATPase subunit; E; isoformV-ATPase; E; 1 isoform; Testis; Subunit assembly

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells by Vian Azzu; Charles Affourtit; Eamon P. Breen; Nadeene Parker; Martin D. Brand (pp. 1378-1383).

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0–4.4 ng/million cells (2.7–14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.

Keywords: Abbreviations; ANT; adenine nucleotide translocase; GSIS; glucose-stimulated insulin secretion; FCS; fetal calf serum; Q; glutamine; ROS; reactive oxygen species; UCP; uncoupling protein; rUCP2; recombinant UCP2Glutamine; Half-life; Mitochondria; Pancreatic beta-cell; Serum starvation; UCP2

Subunit mass fingerprinting of mitochondrial complex I by Nina Morgner; Volker Zickermann; Stefan Kerscher; Ilka Wittig; Albina Abdrakhmanova; Hans-Dieter Barth; Bernhard Brutschy; Ulrich Brandt (pp. 1384-1391).

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5–10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Keywords: Abbreviations; BN-PAGE; blue-native polyacrylamide gel electrophoresis; FMN; flavine-mononucleotide; LILBID; laser induced liquid bead ion desorption; MALDI; matrix-assisted laser desorption/ionisation; MS; mass spectrometry; NADPH; nicotineamide-dinucleotide-phosphate, reduced form; RT-PCR; reverse transcription-polymerase chain reaction; TOF; time of flightLILBID; Membrane protein; Complex I; Mitochondria; Mass spectrometry; Yarrowia lipolytica

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BBA - Bioenergetics (v.1777, #10)