|
|
BBA - Bioenergetics (v.1797, #3)
Mechanism of strong quenching of photosystem II chlorophyll fluorescence under drought stress in a lichen, Physciella melanchla, studied by subpicosecond fluorescence spectroscopy
by Masayuki Komura; Atsushi Yamagishi; Yutaka Shibata; Ikuko Iwasaki; Shigeru Itoh (pp. 331-338).
The mechanism of the severe quenching of chlorophyll (Chl) fluorescence under drought stress was studied in a lichen Physciella melanchla, which contains a photobiont green alga, Trebouxia sp., using a streak camera and a reflection-mode fluorescence up-conversion system. We detected a large 0.31 ps rise of fluorescence at 715 and 740 nm in the dry lichen suggesting the rapid energy influx to the 715–740 nm bands from the shorter-wavelength Chls with a small contribution from the internal conversion from Soret bands. The fluorescence, then, decayed with time constants of 23 and 112 ps, suggesting the rapid dissipation into heat through the quencher. The result confirms the accelerated 40 ps decay of fluorescence reported in another lichen (Veerman et al., 2007 [36]) and gives a direct evidence for the rapid energy transfer from bulk Chls to the longer-wavelength quencher. We simulated the entire PS II fluorescence kinetics by a global analysis and estimated the 20.2 ns−1 or 55.0 ns−1 energy transfer rate to the quencher that is connected either to the LHC II or to the PS II core antenna. The strong quenching with the 3–12 times higher rate compared to the reported NPQ rate, suggests the operation of a new type of quenching, such as the extreme case of Chl-aggregation in LHCII or a new type of quenching in PS II core antenna in dry lichens.
Keywords: Abbreviations; Chl; chlorophyll; DAS; decay-associated spectrum; EET; excitation energy transfer; F; 0; the fluorescence level in the dark adapted state; F; M; the fluorescence level after the saturating pulse illumination; IRF; instrument response function; LHC II; light-harvesting chlorophyll; a; /; b; binding complex II; NPQ; nonphotochemical quenching; PS; photosystem; Q; d; a quencher of excitation energy induced in the dry lichen; RC; reaction center; t; c; time constantChlorophyll fluorescence; Drought stress; Photosynthesis; Photosystem II; Excitation energy transfer; Lichen; Fluorescence quenching; Up-conversion spectroscopy
Low-temperature electron transfer suggests two types of QA in intact photosystem II
by Han Bao; Chunxi Zhang; Yanan Ren; Jingquan Zhao (pp. 339-346).
The correlation between the reduction of QA and the oxidation of TyrZ or Car/ChlZ/Cytb559 in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g=1.95–1.86 QA-•EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from QA-•, Car+•/Chl+• and oxidized Cytb559, while enhancing the amplitude of the S1TyrZ• EPR signal in the intact PSII sample. The quantification and stability of the g=1.95–1.86 EPR signal and signals arising from the oxidized TyrZ and the side-path electron donors, respectively, indicate that the EPR-detectable g=1.95–1.86 QA-• signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/ChlZ/Cytb559), but not of TyrZ. These results imply that two types of QA-• probably exist in the intact PSII sample. The structural difference and possible function of the two types of QA are discussed.
Keywords: Abbreviations; Chl; chlorophyll; Chl; Z; side-path redox active Chl; Car; redox active β-carotene; Cyt; b559; cytochrome b559; D1; D2, reaction center core proteins; DMSO; dimethyl sulfoxide; EDTA; ethylenediaminetetraacetic acid; EPR; electron paramagnetic resonance; MES; 4-morpholine ethanesulfonic acid; P; 680; primary electron donor of PSII; P; D1; P; D2; , two monomeric Chls of P; 680; associated with D; 1; and D; 2; , respectively; Pheo; pheophytin; PPBQ; phenyl-; p; -benzoquinone; PSII; photosystem II; PQ; plastoquinone; PQH; 2; plastoquinol; Q; A; and Q; B; primary and secondary quinone electron acceptors, respectively; Tyr; D; tyrosine 161 of the D; 1; protein; Tyr; Z; tyrosine 160 of the D; 2; proteinPhotosystem II; Q; A; Tyr; Z; Side-path electron donor; Electron transfer; EPR
Redox potential of the Rieske iron–sulfur protein
by Andrey M. Kuznetsov; Ekaterina M. Zueva; Alexei N. Masliy; Lev I. Krishtalik (pp. 347-359).
Quantum-chemical study of structures, energies, and effective partial charge distribution for several models of the Rieske protein redox center is performed in terms of the B3LYP density functional method in combination with the broken symmetry approach using three different atomic basis sets. The structure of the redox complex optimized in vacuum differs markedly from that inside the protein. This means that the protein matrix imposes some stress on the active site resulting in distortion of its structure. The redox potentials calculated for the real active site structure are in a substantially better agreement with the experiment than those calculated for the idealized structure. This shows an important role of the active site distortion in tuning its redox potential. The reference absolute electrode potential of the standard hydrogen electrode is used that accounts for the correction caused by the water surface potential. Electrostatic calculations are performed in the framework of the polarizable solute model. Two dielectric permittivities of the protein are employed: the optical permittivity for calculation of the intraprotein electric field, and the static permittivity for calculation of the dielectric response energy. Only this approach results in a reasonable agreement of the calculated and experimental redox potentials.
Keywords: Abbreviations; B3LYP; gradient-corrected functional described by a combination of Becke's three parameter (B3) hybrid exchange functional and the Lee–Yang–Parr (LYP) correlation functional; BS; broken symmetry approach; CD; circular dichroism; DFT; density functional theory; ESP charges; atomic charges fitted from the molecular electrostatic potential; ExtC; extended model complex; emf; electromotive force; ISP; iron–sulfur protein; MinC; minimal model complex; pdb; Protein Data Bank identifier; SCF; self-consistent field; S.H.E.; standard hydrogen electrode; 6-31G(d,p); standard split-valence atomic basis set including polarization functions on all the atoms; 6-31++G(d,p); the same supplemented by diffuse Gaussian components on all the atoms; TZVP; triple-zeta split-valence basis set supplemented by polarization functions; UB3LYP; spin-unrestricted version of B3LYP; WT; wild typeRieske protein; Redox potential; Protein's dielectric permittivity; Density functional calculation; Broken symmetry approach; Absolute electrode potential
Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex
by Edward A. Berry; Li-shar Huang; Dong-Woo Lee; Fevzi Daldal; Kazuo Nagai; Nobuko Minagawa (pp. 360-370).
Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia ( Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc1 complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Qi site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Qo site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A3 in a fashion similar to the Qo site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Qi and the Qo sites of the fungal cytochrome bc1 complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron–sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc1 complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Qo and Qi sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc1 complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Qi and Qo sites. Moreover, crystal structure of chicken cytochrome bc1 complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Qi and Qo sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc1 inhibitor that acts at both of the active sites of this enzyme.
Keywords: Abbreviations; HQNO or NQNO; heptyl or nonyl hydroxyquinoline-; N; -oxide; nQNO; HQNO or NQNO; DB; 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; DBH; 2; reduced DB; NHDBT; nonylhydroxydioxobenzothiazole; MCLA; 2-methyl-6-(; p; -methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one; MOPS; 3-(; N; -morpholino)propanesulfonic acid; MES; 2-morpholino ethanesulfonateAscochlorin; Cytochrome; bc; 1; complex; Quinol analog inhibitor; Q; o; site; Q; i; site
Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions
by David C. Unitt; Veronica S. Hollis; Miriam Palacios-Callender; Nanci Frakich; Salvador Moncada (pp. 371-377).
We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O2) conditions. The system measures the concentrations of O2 and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O2 concentration and electron turnover of the enzyme. At a high O2 concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration ofl-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O2. At low O2 (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O2 consumption. At both high and low O2 concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.
Keywords: Cytochrome; c; oxidase; Electron turnover; Nitric oxide inactivation; Redox state; Steady-state oxygen
Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium
by Dmitri R. Davydov; Elena V. Sineva; Srinivas Sistla; Nadezhda Y. Davydova; Daniel J. Frank; Stephen G. Sligar; James R. Halpert (pp. 378-390).
We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4–ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4–ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55±6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4–BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.
Keywords: Cytochrome P450 3A4; Flavin domain of cytochrome P450BM-3; Kinetics of electron transfer; Spin equilibrium; Nanodisc; Liposome; Oligomerization; Lifetime; FRET; BODIPY; Cysteine-depleted mutant
Chloroplastic oxidative burst induced by tenuazonic acid, a natural photosynthesis inhibitor, triggers cell necrosis in Eupatorium adenophorum Spreng
by Shiguo Chen; Chunyan Yin; Sheng Qiang; Fenyan Zhou; Xinbin Dai (pp. 391-405).
Tenuazonic acid (TeA), a nonhost-specific phytotoxin produced by Alternaria alternata, was determined to be a novel natural photosynthesis inhibitor owning several action sites in chloroplasts. To further elucidate the mode of its action, studies were conducted to assess the production and involvement of reactive oxygen species (ROS) in the toxic activity of TeA. A series of experiments indicated that TeA treatment can induce chloroplast-derived ROS generation including not only1O2 but also O2−, H2O2 andOH in Eupatorium adenophorum mesophyll cells, resulting from electron leakage and charge recombination in PSII as well as thylakoid overenergization due to inhibition of the PSII electron transport beyond QA and the reduction of end acceptors on the PSI acceptor side and chloroplast ATPase activity. The initial production of TeA-induced ROS was restricted to chloroplasts and accompanied with a certain degree of chloroplast damage. Subsequently, abundant ROS were quickly dispersed throughout whole cell and cellular compartments, causing a series of irreversible cellular harm such as chlorophyll breakdown, lipid peroxidation, plasma membrane rupture, chromatin condensation, DNA cleavage, and organelle disintegration, and finally resulting in rapid cell destruction and leaf necrosis. These results show that TeA causing cell necrosis of host-plants is a result of direct oxidative damage from chloroplast-mediated ROS eruption.
Keywords: TeA; Chloroplast; ROS; Mode of action; Phytotoxin; Photosynthesis inhibitor
De novo design of a non-natural fold for an iron–sulfur protein: Alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core
by Joanna Grzyb; Fei Xu; Lev Weiner; Eduard J. Reijerse; Wolfgang Lubitz; Vikas Nanda; Dror Noy (pp. 406-413).
Using a ‘metal-first’ approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe4S4) cluster protein with a non-natural α-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe4S4 cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron–sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe4S4 clusters in vitro. UV–Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, α-helical coiled-coil binding a single Fe4S4 cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe4S4]+ (S=1/2), with g values of g z, y=(1.970, 1.975), and g x=2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe4S4]+ clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as “wires” for coupling any given set of redox enzymes.
Keywords: Iron sulfur cluster; Protein de novo design; Redox enzyme; Coiled-coil; Four-helix bundle; EPR spectroscopy
The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII)
by Susann Schaller; Dariusz Latowski; Małgorzata Jemioła-Rzemińska; Christian Wilhelm; Kazimierz Strzałka; Reimund Goss (pp. 414-424).
In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.
Keywords: Abbreviations; LHCII; light-harvesting complex of photosystem II; PSII; photosystem II; Vx; violaxanthin; Ax; antheraxanthin; Zx; zeaxanthin; VDE; violaxanthin de-epoxidase; MGDG; monogalactosyldiacylglycerol; DGDG; digalactosyldiacylglycerol; PC; phosphatidylcholine; PG; phosphatidylglycerol; PE; phosphatidylethanolamine; CP; chlorophyll protein complex; NPQ; non-photochemical quenching of chlorophyll a fluorescence; Chl; chlorophyll; H; II; phase; inverted hexagonal phase; DM; n-dodecyl β-D-maltoside; TX-100; Triton X-100; TLC; thin-layer chromatography; SQDG; sulfoquinovosyldiacylglycerol; DES; de-epoxidation state of the violaxanthin cycle pigment poolBilayer lipid; Inverted hexagonal phase; Light-harvesting complex; MGDG; Non-bilayer lipid; Thylakoid membrane; Xanthophyll cycle
Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme
by Tanai Cardona; Ann Magnuson (pp. 425-433).
Cyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N. punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708nm) or phycobilisomes (560nm) specifically. In heterocysts that were pre-illuminated at 708nm, fluorescence from the phycobilisome terminal emitter was observed in the 77K emission spectrum. However, illumination with 560nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750nm, indicating that the 560nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity.
Keywords: Abbreviations; APC; allophycocyanin; ATP; adenosine triphosphate; Chl(s); Chlorophyll(s); EDTA; ethylene diamine tetraacetic acid; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; OCP; orange carotenoid protein; PMSF; phenylmethylsulphonyl fluoride; PSI; Photosystem I; PSII; Photosystem IICyanobacteria; Nostoc; Heterocyst; Phycobilisome; Photosystem I; State transition
|
|
Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases