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BBA - Bioenergetics (v.1777, #5)
Diphenyleneiodonium acutely inhibits reactive oxygen species production by mitochondrial complex I during reverse, but not forward electron transport by Adrian J. Lambert; Julie A. Buckingham; Helen M. Boysen; Martin D. Brand (pp. 397-403).
We investigated the effects of diphenyleneiodonium (DPI) on superoxide production by complex I in mitochondria isolated from rat skeletal muscle. Superoxide production was measured indirectly as hydrogen peroxide production. In a conventional medium containing chloride, DPI strongly inhibited superoxide production by complex I driven by reverse electron transport from succinate. In principle, this inhibition could be explained by an observed decrease in the mitochondrial pH gradient caused by the known chloride–hydroxide antiport activity of DPI. In a medium containing gluconate instead of chloride, DPI did not affect the pH gradient. In this gluconate medium, DPI still inhibited superoxide production driven by reverse electron transport, showing that the inhibition of superoxide production was not dependent on changes in the pH gradient. It had no effect on superoxide production during forward electron transport from NAD-linked substrates in the presence of rotenone (to maximise superoxide production from the flavin of complex I) or antimycin (to maximise superoxide production from complex III), suggesting that the effects of DPI were not through inhibition of the flavin. We conclude that DPI has the novel and potentially very useful ability to prevent superoxide production from the site in complex I that is active during reverse electron transport, without affecting superoxide production during forward electron transport.
Keywords: Mitochondria; Superoxide; Diphenyleneiodonium; Hydrogen peroxide; Reactive oxygen species
Determination of the excitation migration time in Photosystem II by Koen Broess; Gediminas Trinkunas; Arie van Hoek; Roberta Croce; Herbert van Amerongen (pp. 404-409).
The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3±1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5±0.4 ps)−1, the rate of secondary charge separation is (137±5 ps)−1 and the drop in free energy upon primary charge separation is 826±30 cm−1. These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].
Keywords: LHCII; CP29; CP26; CP24; Excitation energy transfer; Non-photochemical quenching
Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae by Christina Kamp; Alexey Silakov; Martin Winkler; Edward J. Reijerse; Wolfgang Lubitz; Thomas Happe (pp. 410-416).
Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C. reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 μmol H2 min−1 mg−1 was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the Hox-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand.
Keywords: [FeFe]-hydrogenase; Sulphur deprivation; Green algae; Purification; EPR
Generation of reactive oxygen species upon strong visible light irradiation of isolated phycobilisomes from Synechocystis PCC 6803 by Sara Rinalducci; Jens Z. Pedersen; Lello Zolla (pp. 417-424).
The mechanism of photodegradation of antenna system in cyanobacteria was investigated using spin trapping ESR spectroscopy, SDS-PAGE and HPLC-MS. Exposure of isolated intact phycobilisomes to illumination with strong white light (3500 μmol m−2 s−1 photosynthetically active radiation) gave rise to the formation of free radicals, which subsequently led to specific protein degradation as a consequence of reactive oxygen species-induced cleavage of the polypeptide backbone. The use of specific scavengers demonstrated an initial formation of both singlet oxygen (1O2) and superoxide (O2−), most likely after direct reaction of molecular oxygen with the triplet state of phycobiliproteins, generated from intersystem crossing of the excited singlet state. In a second phase carbon-based radicals, detected through the appearance of DMPO-R adducts, were produced either via O2− or by direct1O2 attack on amino acid moieties. Thus photo-induced degradation of intact phycobilisomes in cyanobacteria occurs through a complex process with two independent routes leading to protein damage: one involving superoxide and the other singlet oxygen. This is in contrast to the mechanism found in plants, where damage to the light-harvesting complex proteins has been shown to be mediated entirely by1O2 generation.
Keywords: Singlet oxygen; Superoxide; Protein degradation; Phycobiliprotein; ESR spectroscopy; High light stress; Cyanobacteria
Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants by Mikko Tikkanen; Markus Nurmi; Marjaana Suorsa; Ravi Danielsson; Fikret Mamedov; Stenbjörn Styring; Eva-Mari Aro (pp. 425-432).
Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI–LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI–LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.
Keywords: LHCII; Photosystem I; Photosystem II; Photosynthesis; Thylakoid protein phosphorylation; State transition
Feedback regulation of photosynthetic electron transport by NADP(H) redox poise by Simon Hald; Beena Nandha; Patrick Gallois; Giles N. Johnson (pp. 433-440).
When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process — feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco ( Nicotiana tabacum) lines — one having reduced levels of ferredoxin NADP+-reductase (FNR), the enzyme responsible for reducing NADP+, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.
Keywords: Abbreviations; as; antisense; cyt; cytochrome; ΔpH; pH gradient across thylakoid membrane; FNR; ferredoxin NADP; +; -reductase; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; NADP; +; oxidised nicotinamide adenine dinucleotide phosphate; NADPH; reduced nicotinamide adenine dinucleotide phosphate; NADP(H); total pool of NADP; +; and NADPH; NPQ; non photochemical quenching; NPQ; f; fast relaxing NPQ; NPQ; s; slow-relaxing NPQ; PSI; photosystem I; PSII; photosystem II; ROS; reactive oxygen speciesElectron transport; Regulation; Antisense plant; Stress
The energetics of the primary proton transfer in bacteriorhodopsin revisited: It is a sequential light-induced charge separation after all by Sonja Braun-Sand; Pankaz K. Sharma; Zhen T. Chu; Andrei V. Pisliakov; Arieh Warshel (pp. 441-452).
The light-induced proton transport in bacteriorhodopsin has been considered as a model for other light-induced proton pumps. However, the exact nature of this process is still unclear. For example, it is not entirely clear what the driving force of the initial proton transfer is and, in particular, whether it reflects electrostatic forces or other effects. The present work simulates the primary proton transfer (PT) by a specialized combination of the EVB and the QCFF/PI methods. This combination allows us to obtain sufficient sampling and a quantitative free energy profile for the PT at different protein configurations. The calculated profiles provide new insight about energetics of the primary PT and its coupling to the protein conformational changes. Our finding confirms the tentative analysis of an earlier work (A. Warshel, Conversion of light energy to electrostatic energy in the proton pump of Halobacterium halobium, Photochem. Photobiol. 30 (1979) 285–290) and determines that the overall PT process is driven by the energetics of the charge separation between the Schiff base and its counterion Asp85. Apparently, the light-induced relaxation of the steric energy of the chromophore leads to an increase in the ion-pair distance, and this drives the PT process. Our use of the linear response approximation allows us to estimate the change in the protein conformational energy and provides the first computational description of the coupling between the protein structural changes and the PT process. It is also found that the PT is not driven by twist-modulated changes of the Schiff base's p Ka, changes in the hydrogen bond directionality, or other non-electrostatic effects. Overall, based on a consistent use of structural information as the starting point for converging free energy calculations, we conclude that the primary event should be described as a light-induced formation of an unstable ground state, whose relaxation leads to charge separation and to the destabilization of the ion-pair state. This provides the driving force for the subsequent PT steps.
Keywords: Abbreviations; PTR; Proton transport; PT; Proton transfer; SB; Schiff base; EVB; Empirical valence bond; QCFF/PI; quantum mechanical consistent force field for pi-electron; PDLD/S; protein dipoles–Langevin dipoles; LRF; local reaction field; FEP/US; free energy perturbation/umbrella sampling; TS; transition state; LRA; linear response approximation; QM/MM; quantum mechanics–molecular mechanics; SCAAS; surface constraint all atom solvent; MD; molecular dynamics; NQM; nuclear quantum mechanicalProton transport in protein; QM/MM convergence; Energy storage; p; K; a; in proteins
Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4 by Jun Liu; Terry A. Krulwich; David B. Hicks (pp. 453-461).
A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na+/H+ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na+-resistance on an antiporter-deficient E. coli strain, nor did they confer Na+/H+ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.
Keywords: Type II NADH dehydrogenase; Flavoprotein; Deamino-NADH; NAD(P)H: menadione oxidoreductase; Alkaliphile; Na; +; /H; +; antiporter
Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions by Clemens Reinhold; Sylvia Niczyporuk; Karl Christian Beran; Peter Jahns (pp. 462-469).
The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis ( Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.
Keywords: Abbreviations; Ax; antheraxanthin; DEPS; de-epoxidation state; NPQ; non-photochemical quenching; PSII; photosystem II; Vx; violaxanthin; VxDE; violaxanthin de-epoxidase; Zx; zeaxanthin; ZxE; zeaxanthin epoxidaseCarotenoids; Photosynthesis; Photo-oxidative stress; Xanthophyll cycle; Zeaxanthin epoxidase
Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases by Halley Caixeta de Oliveira; Alfredo Wulff; Elzira Elisabeth Saviani; Ione Salgado (pp. 470-476).
The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2−). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2− generation, besides complex III, stimulated NO degradation. Larger amounts of O2− were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2− production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2− production that favors NO degradation in potato tuber mitochondria.
Keywords: Nitric oxide degradation; External NAD(P)H dehydrogenases; Plant mitochondria; Superoxide anion; Electron leakage; Potato tuber