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BBA - Bioenergetics (v.1817, #5)

Title page/Section Editors (pp. i).

Photo-induced electron transfer in intact cells of Rubrivivax gelatinosus mutants deleted in the RC-bound tetraheme cytochrome: Insight into evolution of photosynthetic electron transport by Vermeglio André Verméglio; Sakiko Nagashima; Jean Alric; Pascal Arnoux; Kenji V.P. Nagashima (pp. 689-696).

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc₁ complex is the High Potential Iron–sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron–sulfur Protein, this connection is achieved by the high potential cytochrome c₈. Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P⁺ and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors.► We deleted two major electron carriers, RC-bound tetraheme and HiPIP, of Rubrivivax gelatinosus. ► These mutants still grow under anaerobic photosynthetic conditions. ► The electron donor to the RC is HiPIP in the mutant deleted in RC-bound tetraheme. ► The electron donor to the reaction center is the cytochrome c8 in the double mutant.

Keywords: Abbreviations; B.; Blastochloris; cyt; cytochrome; HiPIP; High Potential Iron–sulfur Protein; HP; high potential; LP; low potential; RC; reaction center; R.; Rhodobacter; R.; RubrivivaxPhotosynthetic bacteria; Rubrivivax gelatinosus; HIPIP; cyt; c; ₈; electron transfer

Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups by Annamaria Tonazzi; Lara Console; Nicola Giangregorio; Cesare Indiveri; Ferdinando Palmieri (pp. 697-704).

The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [³H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.► The hydrophobic binding pocket of the carnitine/acylcarnitine carrier has been studied. ► The protein residues that bind the acyl groups have been identified by mutagenesis. ► Val-25, Pro-78, Val-82, Met-85, Cys-89 bind acyl-moieties of acylcarnitines. ► Ala-276, Ala-279, Cys-283 are involved in other steps of translocation. ► These residues face the protein central cavity forming an hydrophobic half shell.

Keywords: Abbreviations; DTE; dithioerythritol; NEM; N; -ethylmaleimide; Pipes; 1,4-piperazinediethanesulfonic acid; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; WT; wild-type; CAC; carnitine/acylcarnitine carrier; ANC; adenine nucleotide carrier; PIC; phosphate carrier; CIC; citrate carrierCarnitine; Acylcarnitine; Mitochondria; Liposome; Mutagenesis; Transport

Singlet oxygen and non-photochemical quenching contribute to oxidation of the plastoquinone-pool under high light stress in Arabidopsis by Jerzy Kruk; Szymanska Renata Szymańska (pp. 705-710).

The redox state of plastoquinone-pool in chloroplasts is crucial for driving many responses to variable environment, from short-term effects to those at the gene expression level. In the present studies, we showed for the first time that the plastoquinone-pool undergoes relatively fast oxidation during high light stress of low light-grown Arabidopsis plants. This oxidation was not caused by photoinhibition of photosystem II, but mainly by singlet oxygen generated in photosystem II and non-photochemical quenching in light harvesting complex antenna of the photosystem, as revealed in experiments with a singlet oxygen scavenger and with Arabidopsis npq4 mutant. The latter mechanism suppresses the influx of electrons to the plastoquinone-pool preventing its excessive reduction. The obtained results are of crucial importance in light of the function of the redox state of the plastoquinone-pool in triggering many high light-stimulated physiological responses of plants.► Plastoquinone-pool undergoes fast oxidation during high light stress of Arabidopsis. ► This is not caused by photoinhibition of photosystem II. ► Oxidation of the pool is caused by singlet oxygen generated in photosystem II. ► Non-photochemical quenching also contributes to the oxidation.

Keywords: Abbreviations; Chl; chlorophyll; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; DBMIB; 2,5-dibromo-3-methyl-6-isopropyl-; p; -benzoquinone; DPA; diphenylamine; HL; high light; HPLC; high-performance liquid chromatography; PQH; ₂; reduced plastoquinone, plastoquinol; PQ; plastoquinone; PS; photosystem; TNM; tetranitromethane Arabidopsis; High light stress; Plastoquinone; Singlet oxygen; Non-photochemical quenching; Redox state

Singlet oxygen and non-photochemical quenching contribute to oxidation of the plastoquinone-pool under high light stress in Arabidopsis by Jerzy Kruk; Szymanska Renata Szymańska (pp. 705-710).

From red to blue to far-red in Lhca4: How does the protein modulate the spectral properties of the pigments? by Emilie Wientjes; Gemma Roest; Roberta Croce (pp. 711-717).

The first event of photosynthesis is the harvesting of solar energy by a large array of pigments. These pigments are coordinated to proteins that organize them to assure efficient excitation energy transfer. The protein plays an essential role in tuning the spectroscopic properties of the pigments, by determining their site energy and/or by favoring pigment–pigments interactions. Here we investigate how the protein modulates the pigment properties by using a single-point-mutation approach. We monitor changes in the low-energy absorption/emission band of Lhca4, which is well separated from the bulk absorption and thus represents an attractive model system. Moreover, it was recently shown that Lhca4 exists in at least two conformations, a dominating one emitting at 720nm and a second one emitting at 685nm (Kruger et al. PNAS 2011). Here we show that a single amino-acid substitution (from Asn to Gln, which are both chlorophyll-binding residues and only differ for one C―C bond), moves the equilibrium almost completely towards the 685-nm conformation. This indicates that small changes in the protein can have a large effect on the properties of the pigments. We show that His99, which was suggested to coordinate a red-absorbing chlorophyll (Melkozernov and Blankenship, JBC 2003), is not a chlorophyll ligand. We also show that single amino-acid substitutions nearby the chlorophylls allow to tune the emission spectrum of the pigments over a wide range of wavelengths and to modulate the excited-state lifetimes of the complex. These findings are discussed in the light of previously proposed non-photochemical quenching models.► The protein modulates the properties of pigments in light-harvesting complexes. ► Obtained Lhca4 mutants showed 45nm blue and 10nm red shifts of the LT emission maxima. ► Lhca4 fluorescence quantum yield and emitting dipole strengths were modulated. ► The results are discussed in the light of non-photochemical quenching mechanisms.

Keywords: Abbreviations; CT; Charge-transfer; Chl; Chlorophyll; DAS; decay associated spectra; Lhc; light-harvesting complex; Lhca; Light-harvesting complex of Photosystem I; Lhcb; Light-harvesting complex of Photosystem II; Photosystem; PS; RC; reaction centre; TCSPC; time-correlated single photon countingPhotosynthesis; Light-harvesting complex; lhca4; Non-photochemical quenching; Charge-transfer state; Tuning of spectroscopic properties

From red to blue to far-red in Lhca4: How does the protein modulate the spectral properties of the pigments? by Emilie Wientjes; Gemma Roest; Roberta Croce (pp. 711-717).

Preservation of NADH ubiquinone-oxidoreductase activity by Src kinase-mediated phosphorylation of NDUFB10 by Etienne Hebert-Chatelain; Caroline Jose; Nicolas Gutierrez Cortes; Jean-William Dupuy; Christophe Rocher; Jeanne Dachary-Prigent; Thierry Letellier (pp. 718-725).

The tyrosine kinase Src is upregulated in several cancer cells. In such cells, there is a metabolic reprogramming elevating aerobic glycolysis that seems partly dependent on Src activation. Src kinase was recently shown to be targeted to mitochondria where it modulates mitochondrial bioenergetics in non-proliferative tissues and cells. The main goal of our study was to determine if increased Src kinase activity could also influence mitochondrial metabolism in cancer cells (143B and DU145 cells). We have shown that 143B and DU145 cells produce most of the ATP through glycolysis but also that the inhibition of OXPHOS led to a significant decrease in proliferation which was not due to a decrease in the total ATP levels. These results indicate that a more important role for mitochondria in cancer cells could be ensuring mitochondrial functions other than ATP production. This study is the first to show a putative influence of intramitochondrial Src kinase on oxidative phosphorylation in cancer cells. Indeed, we have shown that Src kinase inhibition led to a decrease in mitochondrial respiration via a specific decrease in complex I activities (NADH–ubiquinone oxidoreductase). This decrease is associated with a lower phosphorylation of the complex I subunit NDUFB10. These results suggest that the preservation of complex I function by mitochondrial Src kinase could be important in the development of the overall phenotype of cancer.► The oncogene Src kinase can regulate oxidative phosphorylation in healthy cells. ► We tested if Src kinase can regulate oxidative phosphorylation in cancer cells. ► Mitochondrial Src kinase of cancer cells (143B and DU145) modulates bioenergetics. ► Mitochondrial Src kinase contributes to metabolic profile of cancer cells.

Keywords: Cancer; Src kinase; Phosphorylation; Mitochondria; Oxidative phosphorylation; NDUFB10

Dynamic water networks in cytochrome cbb₃ oxidase by Vivek Sharma; Wikstrom Mårten Wikström; Ville R.I. Kaila (pp. 726-734).

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327–330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb₃. In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b₃ both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b₃. Protonation of Glu323 provides a mechanism to tune the redox potential of heme b₃ with possible implications for proton pumping.► Water networks in cytochrome cbb₃ were studied by MD simulations. ► Water wires connected Glu323 with both P- and N-sides of the membrane. ► The proton affinity of Glu323 increased upon reduction of the active site. ► Water wires for proton translation and oxygen reduction chemistry were observed.

Keywords: Abbreviations; HCO; heme-copper oxidase; MD; molecular dynamics; PLS; proton-loading site; C; c; O; cytochrome; c; oxidase; DFT; density functional theory; C; c; P; cytochrome; c; peroxidase; pT; proton-transferProton channel; Proton pumping; cbb; ₃; -type cytochrome; c; oxidase; Continuum electrostatics; Density functional theory (DFT); Molecular dynamics (MD) simulation

Dynamic water networks in cytochrome cbb₃ oxidase by Vivek Sharma; Wikstrom Mårten Wikström; Ville R.I. Kaila (pp. 726-734).

Polyamines induce aggregation of LHC II and quenching of fluorescence in vitro by Theodoros Tsiavos; Nikolaos E. Ioannidis; Kiriakos Kotzabasis (pp. 735-743).

Dissipation of excess excitation energy within the light-harvesting complex of Photosystem II (LHC II) is a main process in plants, which is measured as the non-photochemical quenching of chlorophyll fluorescence or qE. We showed in previous works that polyamines stimulate qE in higher plants in vivo and in eukaryotic algae in vitro. In the present contribution we have tested whether polyamines can stimulate quenching in trimeric LHC II and monomeric light-harvesting complex b proteins from higher plants. The tetramine spermine was the most potent quencher and induced aggregation of LHC II trimers, due to its highly cationic character. Two transients are evident at 100μM and 350μM for the fluorescence and absorbance signals of LHC II respectively. On the basis of observations within this work, some links between polyamines and the activation of qE in vivo is discussed.► Polyamines quench Chl fluorescence in monomeric Lhcb proteins and trimeric LHC II. ► The relative rank of effectiveness is Spm > Spd > Put. ► Spermine induces aggregation of trimeric LHC II. ► Two transients are evident for the fluorescence and absorbance signals of LHC II.

Keywords: Abbreviations; LHC II; light-harvesting complex II; Chl; chlorophyll; Neo; neoxanthin; Vio; violaxanthin; PAs; polyamines; Spm; spermine; Spd; spermidine; Put; putrescine; ΔpH; thylakoid proton gradient; qE; ΔpH-dependent quenching of chlorophyll fluorescence; DM; dodecyl-β-; d; -maltoside; q; _F; fluorescence quenchingqE; Light harvesting complex II; Aggregation; Fluorescence quenching; Spermine

Polyamines induce aggregation of LHC II and quenching of fluorescence in vitro by Theodoros Tsiavos; Nikolaos E. Ioannidis; Kiriakos Kotzabasis (pp. 735-743).

‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity by Weisova Petronela Weisová; Ujval Anilkumar; Caitriona Ryan; Caoimhín G. Concannon; Jochen H.M. Prehn; Manus W. Ward (pp. 744-753).

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca2⁺ uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100nM FCCP for 30min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5min of the uncoupling stimulus and elevated up to 1h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.

Keywords: Abbreviations; AICAR; 5-amino-4-imidazolecarboxamide riboside; AMP; 5′-adenosine monophosphate; AMPK; 5′-adenosine monophosphate-activated protein kinase; ANOVA; analysis of variance; ATP; adenosine 5′-(tetrahydrogen triphosphate); BSA; bovine serum albumin; Ca; 2; ⁺; calcium; CGNs; cerebellar granule neurons; DIV; days; in vitro; FCCP; carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GLUT 1; glucose transporter 1; GLUT 3; glucose transporter 3; HEPES; N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]; HK-II; hexokinase II; HRP; horse radish peroxidase; kDa; kilodaltons; ∆ψ; _m; mitochondrial membrane potential; NAD; ⁺; /NADH; nicotinamide adenine dinucleotide; NMDA; N; -methyl-; d; -aspartate; NO; nitric oxide; PFK-2; phosphofructokinase-2; PBS; phosphate buffered saline; RNA; ribonucleic acid; ROS; reactive oxygen species; SEM; standard error of the mean; TMRM; tetramethylrhodamine methylester; UCP; uncoupling proteinMitochondrial bioenergetics; Uncoupling; AMPK; ATP; Excitotoxicity; Neuronal preconditioning

Alterations in photosynthetic pigments and amino acid composition of D1 protein change energy distribution in photosystem II by Makio Yokono; Tatsuya Tomo; Ryo Nagao; Hisashi Ito; Ayumi Tanaka; Seiji Akimoto (pp. 754-759).

The marine cyanobacterium Prochlorococcus marinus accumulates divinyl chlorophylls instead of monovinyl chlorophylls to harvest light energy. As well as this difference in its chromophore composition, some amino acid residues in its photosystem II D1 protein were different from the conserved amino acid residues in other photosynthetic organisms. We examined PSII complexes isolated from mutants of Synechocystis sp. PCC 6803, in which chromophore and D1 protein were altered (Hisashi Ito and Ayumi Tanaka, 2011) to clarify the effects of chromophores/D1 protein composition on the excitation energy distribution. We prepared the mutants accumulating divinyl chlorophyll (DV mutant). The amino acid residues of V205 and G282 in the D1 protein were substituted with M205 and C282 in the DV mutant to mimic Prochlorococcus D1 protein (DV-V205M/G282C mutant). Isolated PSII complexes were analyzed by time-resolved fluorescence spectroscopy. Energy transfer in CP47 was interrupted in PSII containing divinyl chlorophylls. The V205M/G282C mutation did not recover the energy transfer pathway in CP47, instead, the mutation allowed the excitation energy transfer from CP43 to CP47, which neighbors in the PSII dimer. Mutual orientation of the subcomplexes of PSII might be affected by the substitution. The changes of the energy transfer pathways would reduce energy transfer from antennae to the PSII reaction center, and allow Prochlorococcus to acquire light tolerance.► We analyzed energy transfer in PSII containing 3,8-divinyl chlorophyll a (DV-Chl). ► Steady-state and time-resolved fluorescence spectroscopies were used. ► Energy transfer was interrupted in CP47 containing DV-Chl. ► Unique amino acids residues in Prochlorococcus interfered energy transfer from CP43 to D1. ► The unique residues accelerated energy transfer from CP43 to CP47 in the PSII dimer.

Keywords: Abbreviations; PSII; photosystem II; RC; reaction center; MV-Chl; chlorophyll; a; DV-Chl; 3,8-divinyl chlorophyll; a; FDAS; fluorescence decay-associated spectrum; TRFS; time-resolved fluorescence spectraDivinyl chlorophyll; Photosystem II; Delayed fluorescence; Cyanobacteria

On the relationship between non-photochemical quenching and photoprotection of Photosystem II by Petar H. Lambrev; Yuliya Miloslavina; Peter Jahns; Alfred R. Holzwarth (pp. 760-769).

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. NPQ is typically characterized by modulated pulse fluorometry and it is often assumed implicitly to be a good proxy for the actual physiological photoprotection capacity of the organism. Using the results of previously published ultrafast fluorescence measurements on intact leaves of w.t. and mutants of Arabidopsis (Holzwarth et al. 2009) we have developed exact relationships for the fluorescence quenching and the corresponding Photosystem II acceptor side photoprotection effects under NPQ conditions. The approach based on the exciton–radical pair equilibrium model assumes that photodamage results from triplet states generated in the reaction center. The derived relationships allow one to distinguish and determine the individual and combined quenching as well as photoprotection contributions of each of the multiple NPQ mechanisms. Our analysis shows inter alia that quenching and photoprotection are not linearly related and that antenna detachment, which can be identified with the so-called qE mechanism, contributes largely to the measured fluorescence quenching but does not correspond to the most efficient photoprotective response. Conditions are formulated which allow simultaneously the maximal photosynthetic electron flow as well as maximal acceptor side photoprotection. It is shown that maximal photoprotection can be achieved if NPQ is regulated in such a way that PSII reaction centers are open under given light conditions. The results are of fundamental importance for a proper interpretation of the physiological relevance of fluorescence-based NPQ data.► Photoprotection processes in Photosystem II were simulated using a kinetic model. ► The change in fluorescence and chlorophyll triplet yield was calculated. ► Non-photochemical quenching and photoprotection are not linearly related. ► Photoprotection by antenna quenching is more effective than antenna detachment. ► Maximal photoprotection is achieved when reaction centers are kept open.

Keywords: Non-photochemical quenching; Arabidopsis; Photosystem II; Mathematical modeling

On the relationship between non-photochemical quenching and photoprotection of Photosystem II by Petar H. Lambrev; Yuliya Miloslavina; Peter Jahns; Alfred R. Holzwarth (pp. 760-769).

Keywords: Non-photochemical quenching; Arabidopsis; Photosystem II; Mathematical modeling

The chl a fluorescence intensity is remarkably insensitive to changes in the chlorophyll content of the leaf as long as the chl a/ b ratio remains unaffected by Dinc Emine Dinç; M. Georgina Ceppi; Toth Szilvia Z. Tóth; Sándor Bottka; Gert Schansker (pp. 770-779).

The effects of changes in the chlorophyll (chl) content on the kinetics of the OJIP fluorescence transient were studied using two different approaches. An extensive chl loss (up to 5-fold decrease) occurs in leaves suffering from either an Mg²⁺ or SO₄²⁻ deficiency. The effects of these treatments on the chl a/ b ratio, which is related to antenna size, were very limited. This observation was confirmed by the identical light intensity dependencies of the K, J and I-steps of the fluorescence rise for three of the four treatments and by the absence of changes in the F_{685 nm}/ F_{695 nm}-ratio of fluorescence emission spectra measured at 77K. Under these conditions, the F₀ and F_M-values were essentially insensitive to the chl content. A second experimental approach consisted of the treatment of wheat leaves with specifically designed antisense oligodeoxynucleotides that interfered with the translation of mRNA of the genes coding for chl a/ b binding proteins. This way, leaves with a wide range of chl a/ b ratios were created. Under these conditions, an inverse proportional relationship between the F_M values and the chl a/ b ratio was observed. A strong effect of the chl a/ b ratio on the fluorescence intensity was also observed for barley Chlorina f2 plants that lack chl b. The data suggest that the chl a/ b ratio (antenna size) is a more important determinant of the maximum fluorescence intensity than the chl content of the leaf.► Mg²⁺ and sulfur deficiencies: lower leaf chl content, no effect on antenna size. ► Changes in leaf chl content (up to 80%) hardly affect F₀ and F_M. ► Antisense ODNs targeted to cab-mRNA can slow antenna formation down. ► Differences in antenna size (chl a/ b ratio) have an important effect on F_M. ► The optical properties of the leaf have little effect on the fluorescence kinetics.

Keywords: Abbreviations; C; control; =; complete solution; CAB; chlorophyll; a; /; b; binding protein; chl; chlorophyll; DMF; N,N-dimethylformamide; F; ₀; and; F; _M; fluorescence intensity measured when all photosystem II reaction centers are open or closed respectively; FR; far red; F; _V; variable fluorescence; HS; half-SO; ₄; ²⁻; solution; ODN; oligodeoxynucleotide; OJIP-transient; fluorescence induction transient defined by the names of its intermediate steps:; O; =; 20; μs,; J; =; 3; ms,; I; =; 30; ms and; P; =; the maximum fluorescence intensity; PQ; plastoquinone; PSII and PSI; photosystems II and I, respectively; Q; _A; and Q; _B; primary and secondary quinone electron acceptors of photosystem II respectively; TS; traces of SO; ₄; ²⁻; solution; V; _J; and V; _I; height of the J and I steps as a fraction of the variable fluorescence; WM; without magnesium and half-SO; ₄; ²⁻; solutionChl; a; fluorescence; Mineral deficiency; Chlorophyll content; Antisense oligodeoxynucleotides; Light gradient

An investigation into a cardiolipin acyl chain insertion site in cytochrome c by Badri S. Rajagopal; Gary G. Silkstone; Peter Nicholls; Michael T. Wilson; Jonathan A.R. Worrall (pp. 780-791).

Mitochondrial cytochrome c associates with the phosphoplipid cardiolipin (CL) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion into cytochrome c of an acyl chain, resulting in the dissociation of the axial Met-80 heme-iron ligand. The resulting five coordinate cytochrome c/CL complex has peroxidatic properties leading to peroxidation of CL and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cytochrome c from the mitochondria into the cytoplasm. Two distinct surface regions on cytochrome c have been suggested to mediate CL acyl chain insertion and this study has probed one of these regions. We have constructed a series of alanine mutants aimed at disrupting a surface cleft formed between residues 67–71 and 82–85. The physicochemical properties, peroxidase activity, CL binding, and kinetics of carbon monoxide (CO) binding to the ferrous cytochrome c/CL complex have been assessed for the individual mutants. Our findings reveal that the majority of mutants are capable of binding CL in the same apparent stoichiometry as the wild-type protein, with the extent to which the Met-80 ligand is bound in the ferrous cytochrome c/CL complex being mutant specific at neutral pH. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the CL binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cytochrome c/CL complex.► A putative cardiolipin insertion site on mitochondrial cytochrome c has been studied. ► A series of alanine mutations in cytochrome c have been constructed. ► Cardiolipin interaction has been assessed using carbon monoxide as a small ligand probe. ► The alanine mutations influence the extent of CO binding in the ferrous cytochrome c complex. ► Arg-91 alters the cardiolipin binding stoichiometry.

Keywords: Abbreviations; cyt; c; c; ytochrome; c; CL; cardiolipin; ET; electron transfer; WT; wild-type; Amp; ampicillan; CD; circular dichroism; GuHCl; guanidine hydrochloride; CAPS; 3-(cyclohexylamino)-1-propanesulfonic acid; Tris; tris(hydroxymethyl)aminomethane; E; _m; midpoint redox potential; TMPD; N; ,; N; ,; N; ',; N; '-tetramethyl-; p; -phenylenediamineCytochrome; c; Phospholipids; Cardiolipin; Apoptosis; Peroxidase; Ligand binding

An investigation into a cardiolipin acyl chain insertion site in cytochrome c by Badri S. Rajagopal; Gary G. Silkstone; Peter Nicholls; Michael T. Wilson; Jonathan A.R. Worrall (pp. 780-791).

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803 by Qing Jun Wang; Abhay Singh; Hong Li; Ladislav Nedbal; Louis A. Sherman; Govindjee; John Whitmarsh (pp. 792-801).

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H₂O and depends on photosystem I (PSI) to reduce NADP⁺. Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O₂ evolution in vivo. The net light-induced O₂ evolution requires glucose and can be sustained for more than 30min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O₂ evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b₆f complex. Moreover, both O₂ evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using¹⁴C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO₂ in the light compared to the dark. However, the rate of the light-minus-dark CO₂ assimilation accounts for just over half of the net light-induced O₂ evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O₂ evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO₂ assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme.► Synechocystis mutants lacking Photosystem I are able to produce oxygen in light. ► This occurs in mixotrophically grown cells and in the presence of glucose. ► This oxygen evolution requires Photosystem II and plastoquinone pool. ► A KCN-sensitive pathway exists for this oxygen evolution to occur. ► It is accompanied by low CO₂ assimilation; an alternate pathway must exist.

Keywords: Abbreviations; Chl; chlorophyll; PSII; photosystem II; Cyt bf; cytochrome b; ₆; f; Cyt c; ₆; cytochrome c; ₆; PSI; photosystem I; Fd; ferredoxin; FNR; ferredoxin-NADP; ⁺; -oxidoreductase; Pheo; pheophytin; FQR; ferredoxin-plastoquinone-oxidoreductase; PQ; plastoquinone; PQH; ₂; plastoquinol; P680; special pair of Chl; a; molecules for primary photochemistry in PSII; P700; special pair of Chl; a; molecules for primary photochemistry in PSI; Q; _A; first plastoquinone electron acceptor of PSII; Q; _B; second plastoquinone electron acceptor of PSII; Q; _o; the plastoquinol oxidase site of the Cyt bf complex; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; KCN; potassium cyanide; FeCN; potassium ferricyanide; DCBQ; 2,6-dichloro-; p; -benzoquinone; DBMIB; 2,5-dibromo-3-methyl-6-isopropyl-; p; -benzoquinone; DCPIP; 2,6-dichlorophenolindophenol; MV; methyl viologen; WT; wild type; ΔPSI; PSI deletion; PEP; phosphoenol pyruvate; Rubisco; ribulose-1,5-bisphosphate carboxylase/oxygenasePhotosynthesis; Electron transport; CO; ₂; assimilation; Photosystem I; Oxygen evolution; Synechocystis

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide by Alain Boussac; Naoko Ishida; Miwa Sugiura; Fabrice Rappaport (pp. 802-810).

The active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S₀ to S₄) before O₂ is evolved. It consists of a Mn₄CaO₅ cluster and Tyr_Z, a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7Å away from the Mn₄CaO₅ cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I⁻ for Cl⁻ in the cyanobacterium Thermosynechococcus elongatus with either Ca2⁺ or Sr2⁺ biosynthetically assembled into the Mn₄ cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV–visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl⁻/I⁻ exchange considerably slowed down the two S₃Tyr_Z^•→(S₃Tyr_Z^•)′→S₀ reactions in which the fast phase, S₃Tyr_Z^•→(S₃Tyr_Z^•)′, reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on Tyr_Z^• and the slow phase corresponds to the S₀ and O₂ formations and to a second proton release. The t_1/2 for S₀ formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the Tyr_Z^• reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca2⁺ binding site and the Cl⁻ binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl⁻ and Ca2⁺ binding sites by which proton release may be channelled.Display Omitted► Chloride is substituted for iodide in Photosystem II from T. elongatus. ► Ca/I-PSII and Sr/I-PSII have a high O₂ evolving activity. ► The t_1/2 of S₃TyrZ^•′ to S₀ is 1.1ms in Ca/Cl-PSII and 6ms in Ca/I-PSII. ► The t_1/2 of S₃TyrZ^•′ to S₀ is 4.8ms in Sr/Cl-PSII and 45ms in Sr/I-PSII. ► Ca²⁺ and Cl^– interact via the H-bond and/or H₂O network(s) connecting the 2 sites.

Keywords: Abbreviations; PSII; Photosystem II; Chl; chlorophyll; PPBQ; phenyl-; p; -benzoquinone; MES; 2-(N-morpholino) ethanesulfonic acid; P; ₆₈₀; chlorophyll dimer acting as the second electron donor; Q; _A; primary quinone acceptor; Q; _B; secondary quinone acceptor; 43H; T. elongatus; strain with a His-tag on the C terminus of CP43; EPR; Electron Paramagnetic Resonance; PQ; plastoquinone 9; WT*1, WT*2, WT*3; mutant; T. elongatus; cells expressing only the; psbA1; ,; psbA2; ,; psbA3; gene, respectively; Pheo; _D1; pheophytin; P; _D1; and P; _D2; Chl monomer of P; ₆₈₀; on the D1 or D2 side, respectivelyPhotosystem II; Chloride; Oxygen evolution

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide by Alain Boussac; Naoko Ishida; Miwa Sugiura; Fabrice Rappaport (pp. 802-810).

Tyrosine triad at the interface between the Rieske iron–sulfur protein, cytochrome c₁ and cytochrome c₂ in the bc₁ complex of Rhodobacter capsulatus by John A. Kyndt; John C. Fitch; Robert E. Berry; Matt C. Stewart; Kevin Whitley; Terry E. Meyer; F. Ann Walker; Michael A. Cusanovich (pp. 811-818).

A triad of tyrosine residues (Y152–154) in the cytochrome c₁ subunit (C1) of the Rhodobacter capsulatus cytochrome bc₁ complex (BC1) is ideally positioned to interact with cytochrome c₂ (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations—A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.► We made six mutants of Rhodobacter capsulatus cytochrome c₁. ► Mutant Y152A and Y153F showed no change in redox potential, spectra, or activity. ► Y153Q and Y153A had altered redox potentials and significant loss in activity. ► Close packing of Y153 with P184 is likely to stabilize the sixth heme ligand. ► Mutant Y154A was unstable, but a pseudorevertant arose which regained some activity.

Keywords: Abbreviations; BC1; ubiquinol cytochrome; c; reductase or Rieske iron–sulfur protein/Cytochrome; b; /Cytochrome; c; ₁; complex; C2; cytochrome; c; ₂; ∆fbc; mutant containing a deletion of the whole BC1 operon; RCVB; a minimal growth medium containing mineral salts, essential vitamins, and malate as a carbon source; MOPS; 3-(N-morpholino)-propanesulfonic acid Rhodobacter capsulatus; BC1 complex; Mutagenesis; Redox potential

In Polytomella sp. mitochondria, biogenesis of the heterodimeric COX2 subunit of cytochrome c oxidase requires two different import pathways by Jimenez-Suarez Alejandra Jiménez-Suárez; Vazquez-Acevedo Miriam Vázquez-Acevedo; Rojas-Hernandez Andrés Rojas-Hernández; Soledad Funes; Salvador Uribe-Carvajal; Gonzalez-Halphen Diego González-Halphen (pp. 819-827).

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130–140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.Display Omitted► We developed a method to obtain import-competent mitochondria from Polytomella sp. ► The in vitro import of split COX2 subunits is described for the first time. ► COX2A follows an energy-dependent import pathway to the inner membrane. ► A lower hydrophobicity of the first transmembrane stretch of COX2A is critical. ► COX2B subunit is imported directly into the inter-membrane space.

Keywords: Polytomella; sp.; Chlamydomonas reinhardtii; Mitochondrial targeting signal; COX2; Cytochrome; c; oxidase biogenesis

Evidence for a relationship between mitochondrial Complex I activity and mitochondrial aldehyde dehydrogenase during nitroglycerin tolerance: Effects of mitochondrial antioxidants by Remedios Garcia-Bou; Milagros Rocha; Nadezda Apostolova; Raul Herance; Antonio Hernandez-Mijares; Victor M. Victor (pp. 828-837).

The medical use of nitroglycerin (GTN) is limited by patient tolerance. The present study evaluated the role of mitochondrial Complex I in GTN biotransformation and the therapeutic effect of mitochondrial antioxidants. The development of GTN tolerance (in rat and human vessels) produced a decrease in mitochondrial O₂ consumption. Co-incubation with the mitochondria-targeted antioxidant mitoquinone (MQ, 10−⁶mol/L) or with glutathione ester (GEE, 10−⁴mol/L) blocked GTN tolerance and the effects of GTN on mitochondrial respiration and aldehyde dehydrogenase 2 (ALDH-2) activity. Biotransformation of GTN depended on the mitochondria being functionally active, particularly mitochondrial Complex I. Tolerance induced mitochondrial ROS production and oxidative stress, though these effects were not detected in HUVECρ⁰ cells or Complex I mutant cells. Experiments performed to evaluate Complex I-dependent respiration demonstrated that its inhibition by GTN was prevented by the antioxidants in control samples. These results point to a key role for mitochondrial Complex I in the adequate functioning of ALDH-2. In addition, we have identified mitochondrial Complex I as one of the targets at which the initial oxidative stress responsible for GTN tolerance takes place. Our data also suggest a role for mitochondrial-antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.► Mitochondrial Complex I is undermined following continuous exposure to GTN. ► GTN induces oxidative stress, with mitochondria both its source and its target. ► Complex I plays a role in GTN biotransformation particularly regarding ROS production. ► Complex I can regulate ALDH-2 activity through the availability of NAD⁺. ► Mitochondrial-antioxidants show a therapeutic effect in the control of the tolerance that accompanies chronic nitrate use.

Keywords: Oxidative stress; Nitric oxide; Nitroglycerin; Mitochondria; ALDH-2; Antioxidant

miR-127-5p targets the 3′UTR of human β-F1-ATPase mRNA and inhibits its translation by Imke M. Willers; Martinez-Reyes Inmaculada Martínez-Reyes; Martinez-Diez Marta Martínez-Diez; José M. Cuezva (pp. 838-848).

The mitochondrial H⁺-ATP synthase is a bottleneck component in the provision of metabolic energy by oxidative phosphorylation. The expression of its catalytic subunit (β-F1-ATPase) is stringently controlled at post-transcriptional levels during oncogenesis, the cell cycle and in development. Here we show that miR-127-5p targets the 3′UTR of β-F1-ATPase mRNA (β-mRNA) significantly reducing its translational efficiency without affecting β-mRNA abundance. Despite the reduced expression of β-F1-ATPase in most human carcinomas, we observed no expression of miR-127-5p in different human cancer cell lines, minimizing the potential role of miR-127-5p as a regulator of the bioenergetic activity of mitochondria in cancer. In contrast, miR-127-5p is highly over-expressed in the human fetal liver. Consistent with previous findings in the rat, the expression of β-F1-ATPase in the human liver also seems to be controlled at post-transcriptional levels during development, what might suggest a role for miR-127-5p in controlling β-mRNA translation and thus in defining the bioenergetic activity of human liver mitochondria. Moreover, immunolocalization techniques and subcellular fractionation experiments using different antibodies against β-F1-ATPase reveal that the ectopic expression of β-F1-ATPase at the cell surface of the hepatocytes and HepG2 cells is negligible or stands for scrutiny.Display Omitted► miR-127-5p targets the 3′UTR of human β-F1-ATPase mRNA. ► miR-127-5p inhibits human β-F1-ATPase mRNA translation. ► miR-127-5p is highly over-expressed in the human fetal liver. ► Liver β-F1-ATPase expression is post-transcriptionally regulated during development.

Keywords: Abbreviations; β- α- and γ-F1-ATPase; subunits β, α and γ of the mitochondrial H; ⁺; -ATP synthase; β- and γ-mRNA; mRNAs of subunits β- and γ-F1-ATPase; G3BP1; Ras-GAP SH3 binding protein 1; GFP; green fluorescent protein; SDH-B; succinate dehydrogenase subunit B; 3; ′; UTR; 3; ′; untranslated region; ERRγ and ERRα; estrogen receptor gamma and alpha transcription factors; PGC-1α; Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; ATP5B; β-F1-ATPase geneMitochondria; H; ⁺; -ATP synthase; Liver; Translational control; Human development; Pathology

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BBA - Bioenergetics (v.1817, #5)