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BBA - Bioenergetics (v.1797, #4)
Stimulation of F1-ATPase activity by sodium dodecyl sulfate
by Mohammad Delawar Hossain; Shou Furuike; Yasuhiro Onoue; Kengo Adachi; Masasuke Yoshida; Kazuhiko Kinosita Jr. (pp. 435-442).
F1-ATPase is a rotary molecular motor in which the γ subunit rotates inside the cylinder made of α3β3 subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F1-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2mM ATP. Maximal stimulation was obtained at 0.003% (w/v) SDS, the initial (least inhibited) activity being about 1.4 times and the steady-state activity 3–4 times the values in the absence of SDS. Rotation rates observed with a 40-nm gold bead or a 0.29-μm bead duplex as well as the torque were unaffected by the presence of 0.003% SDS. The fraction of beads that rotated, in contrast, tended to increase in the presence of SDS. SDS seems to bring inactive F1 molecules into an active form but it does not alter or enhance the function of already active F1 molecules significantly.
Keywords: Single molecule; Optical microscopy; Detergent; Torque; ATP synthase; ATP hydrolysis
Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei
by Yasutoshi Kido; Kimitoshi Sakamoto; Kosuke Nakamura; Michiyo Harada; Takashi Suzuki; Yoshisada Yabu; Hiroyuki Saimoto; Fumiyuki Yamakura; Daijiro Ohmori; Anthony Moore; Shigeharu Harada; Kiyoshi Kita (pp. 443-450).
The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis–Menten kinetics ( Km of 338μM and Vmax of 601μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.
Keywords: Abbreviations; AOX; alternative oxidase; DM; n; -dodecyl-; β; -; d; -maltopyranoside; EPR; electron paramagnetic resonance; ICP-MS; inductively coupled plasma-mass spectrometer; IPTG; isopropyl,; β; -; d; -1-thiogalactoside; k; cat; molecular activity; C10E8; octaethylene glycol-monododecylether; OG; n; -octyl-; β; -; d; -glucopyranoside; rTAO; recombinant trypanosome alternative oxidase; SHAM; salycylhydroxamic acid; TAO; trypanosome alternative oxidase; Ubiquinol; reduced form ubiquinoneAlternative oxidase; Membrane-bound diiron protein; Trypanosoma brucei; Ascofuranone; Chemotherapy
Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II membranes
by Pospišil Pavel Pospíšil; Arjun Tiwari (pp. 451-456).
The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b559 (cyt b559) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HPFe2+) form of cyt b559, whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HPFe3+) form of cyt b559. Light-induced conversion of cyt b559 from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b559 after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b559 proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b559 depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b559 is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.
Keywords: Abbreviations; Capso; N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid; Chl; chlorophyll; cyt; b; 559; cytochorome; b; 559; DEPC; diethylpyrocarbonate; E; m; midpoint redox potential; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HP; Fe3+; high-potential form of cyt; b; 559; with oxidized heme iron; HP; Fe2+; high-potential form of cyt; b; 559; with reduced heme iron; IP; intermediate-potential form of cyt; b; 559; LP; low-potential form of cyt; b; 559; MDGD; monogalactosyldiglycerol; MES; 2-[N-Morpholino]ethanesulfonic acid; PSII; photosystem II; Tricine; N-[tris(hydroxymethyl)methyl]glycine; SQDG; sulfoquinovosyldiacylglycerolCytochorome; b; 559; Photosystem II; Redox potential; Molecular oxygen
The molecular structure of the IsiA–Photosystem I supercomplex, modelled from high-resolution, crystal structures of Photosystem I and the CP43 protein
by Yinan Zhang; Min Chen; W. Bret Church; Kwok Wai Lau; Anthony W.D. Larkum; Lars S. Jermiin (pp. 457-465).
We present the molecular structure of the IsiA–Photosystem I (PSI) supercomplex, inferred from high-resolution, crystal structures of PSI and the CP43 protein. The structure of iron-stress-induced A protein (IsiA) is similar to that of CP43, albeit with the difference that IsiA is associated with 15 chlorophylls (Chls), one more than previously assumed. The membrane-spanning helices of IsiA contain hydrophilic residues many of which bind Chl. The optimal structure of the IsiA–PSI supercomplex was inferred by systematically rearranging the IsiA monomers and PSI trimer in relation to each other. For each of the 6,969,600 structural configurations considered, we counted the number of optimal Chl–Chl connections (i.e., cases where Chl-bound Mg atoms are ≤25Å apart). Fifty of these configurations were found to have optimal energy-transfer potential. The 50 configurations could be divided into three variants; one of these, comprising 36 similar configurations, was found to be superior to the other configurations in terms of its potential to transfer excitation energy to the reaction centres under low-light conditions and its potential to dissipate excess energy under high-light conditions. Compared to the assumed model [Biochemistry 42 (2003) 3180–3188], the new Chl increases by 7% the ability of IsiA to harvest sunlight while the rearrangement of the constituent components of the IsiA–PSI supercomplex increases by 228% the energy-transfer potential. In conclusion, our model allows us to explain how the IsiA–PSI supercomplex may act as an efficient light-harvesting structure under low-light conditions and as an efficient dissipater of excess energy under high-light conditions.
Keywords: IsiA–PSI supercomplex; Chlorophyll; Molecular modelling; Multi-objective optimization; Energy-transfer potential; Photosynthesis
Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis
by Manuela Nilkens; Eugen Kress; Petar Lambrev; Yuliya Miloslavina; Muller Marc Müller; Alfred R. Holzwarth; Peter Jahns (pp. 466-475).
The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10–15min) and relaxation (lifetime 10–15min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.
Keywords: Abbreviations; Ax; antheraxanthin; Chl; chlorophyll; NPQ; non-photochemical quenching; PS II; photosystem II; qE; energy-dependent quenching; qI; photoinhibitory quecnhing; qT; state transition quenching; qZ; zeaxanthin-dependent quenching; Vx; violaxanthin; Zx; zeaxanthinEnergy dissipation; Non-photochemical quenching; Photosynthesis; Xanthophyll cycle; Zeaxanthin
Mitochondrial metabolism during fasting-induced daily torpor in mice
by Jason C.L. Brown; James F. Staples (pp. 476-486).
During fasting, mice ( Mus musculus) undergo daily bouts of torpor, considerably reducing body temperature ( Tb) and metabolic rate (MR). We examined females of different laboratory strains (Balb/c, C57/6N, and CD1) to determine whether liver mitochondrial metabolism is actively reduced during torpor. In all strains, we found that state 3 (phosphorylating) respiration rate measured at 37°C was reduced up to 35% during torpor for at least one of the substrates (glutamate and succinate) used to fuel respiration. The extent of this suppression varied and was correlated with Tb at sampling. This suggests that, at the biochemical level, the transition to and from a hypometabolic torpid state is gradual. In fasted non-torpid animals, Tb and MR still fluctuated greatly: Tb dropped by as much as 4°C and MR was reduced up to 25% compared to fed controls. Changes in Tb and MR in fasted, non-torpid animals were correlated with changes in mitochondrial state 3 respiration rate measured at 37°C. This suggests that fasting mice may conserve energy even when not torpid by occasionally reducing Tb and mitochondrial oxidative capacity to reduce MR. Furthermore, proton conductance was higher in torpid compared to non-torpid animals when measured at 15°C (the lower limit of torpid Tb). This pattern is similar to that reported previously for daily torpor in Phodopus sungorus.
Keywords: Liver; Fasting; Calorie restriction; Proton leak; Top-down regulatory analysis
Conversion of the g=4.1 EPR signal to the multiline conformation during the S2 to S3 transition of the oxygen evolving complex of Photosystem II
by Maria Chrysina; Georgia Zahariou; Nikolaos Ioannidis; Vasili Petrouleas (pp. 487-493).
The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S0–S1,…,S3–(S4)S0 during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)4Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S2 shows a notable heterogeneity with two spectral forms; a g=2 ( S=1/2) multiline, and a g=4.1 ( S=5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S3 they exhibit the same characteristic g=10 ( S=3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g=4.1 conformation during advancement to S3 is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S2 to S3 transition (ca 230K). We observed that, above about 220K the g=4.1 signal converts to a multiline form prior to advancement to S3. We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S2 to S3 transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)4Ca with a low-spin ground state advances to S3.
Keywords: Abbreviations; PSII; Photosystem II; OEC; oxygen evolving complex; S states; S; 0; ,…,S; 4; oxidation states of the OEC; tyr Z or Y; Z; tyrosine 161 of the D2 protein; tyr D or Y; D; tyrosine 160 of the D1 protein; P; 680; the primary electron donor in PSII; Signal II; the EPR signal of Y; D; Metalloradical or split signals; EPR signals in the; g; 2 region attributed to the broadening of the tyr Z; spectrum by a weak magnetic interaction with the Mn cluster; Q; A; , Q; B; the primary, secondary plastoquinone electron acceptors of PSII; MES; 2-[N-Morpholineethanesulfonic acid]; chl; chlorophyll; DCBQ; di-chloro-; p; -benzoquinone; PpBQ; phenyl-; p; -benzoquinone; Duroquinone; tetramethyl benzoquinone; NIR light; near infrared light; EPR; electron paramagnetic resonancePhotosystem II; EPR; Mn cluster; g; =; 4.1; Multiline; Tyrosine Z
X-ray absorption studies of Zn2+-binding sites in Escherichia coli transhydrogenase and its βH91K mutant
by Giulia Veronesi; Simon J. Whitehead; Francesco Francia; Lisa Giachini; Federico Boscherini; Giovanni Venturoli; Nick P.J. Cotton; J. Baz Jackson (pp. 494-500).
Transhydrogenase couples hydride transfer between NADH and NADP+ to proton translocation across a membrane. The binding of Zn2+ to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn2+ bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which βHis91 was replaced by Lys (βH91K). This well-conserved His residue, located in the membrane-spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn2+. The XAFS analysis has identified a Zn2+-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn2+ inhibits proton translocation by competing with H+ binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and βH91K. Evidently, βHis91 is not directly involved in Zn2+ binding. The locus of βHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.
Keywords: Transhydrogenase; XAFS; Zinc-binding site; Metal-ion inhibition; Proton translocation
Photosystem I light-harvesting complex Lhca4 adopts multiple conformations: Red forms and excited-state quenching are mutually exclusive
by Francesca Passarini; Emilie Wientjes; Herbert van Amerongen; Roberta Croce (pp. 501-508).
In this work we have investigated the origin of the multi-exponential fluorescence decay and of the short excited-state lifetime of Lhca4. Lhca4 is the antenna complex of Photosystem I which accommodates the red-most chlorophyll forms and it has been proposed that these chlorophylls can play a role in fluorescence quenching. Here we have compared the fluorescence decay of Lhca4 with that of several Lhca4 mutants that are affected in their red form content. The results show that neither the multi-exponentiality of the decay nor the fluorescence quenching is due to the red forms. The data indicate that Lhca4 exists in multiple conformations. The presence of the red forms, which are very sensitive to changes in the environment, allows to spectrally resolve the different conformations: a “blue” conformation with a short lifetime and a “red” one with a long lifetime. This finding strongly supports the idea that the members of the Lhc family are able to adopt different conformations associated with their light-harvesting and photoprotective roles. The ratio between the conformations is modified by the substitution of lutein by violaxanthin. Finally, it is demonstrated that the red forms cannot be present in the quenched conformation.
Keywords: Abbreviations; PS; Photosystem; Chl; Chlorophyll; DAS; Decay associated spectra; Lhc; Light-harvesting complex; Lhca; Light-harvesting complex of Photosystem I; Lhcb; Light-harvesting complex of Photosystem II; RC; Reaction centre; TRES; Time-resolved emission spectraPhotosynthesis; Antenna complex; Time-resolved fluorescence; Low-energy form; Fluorescence quenching; Mutation analysis
Energy conservation by Rhodothermus marinus respiratory complex I
by Ana P. Batista; Andreia S. Fernandes; Ricardo O. Louro; Julia Steuber; Manuela M. Pereira (pp. 509-515).
A sodium ion efflux, together with a proton influx and an inside-positive ΔΨ, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by23Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.
Keywords: Abbreviations; ACMA; 9-amino-6-chloro-2-methoxyacridine; CCCP; carbonyl cyanide; m; -chlorophenyl hydrazone; DDM; n-dodecyl-β-D-maltoside; DMN; 2,3-Dimethyl-1,4-naphthoquinone; Dy(PPPi); 2; 7; −; dysprosium (III) tripolyphosphate; HiPIP; high-potential iron–sulphur protein; Menadione; 2-methyl-1,4-naphthoquinone; NQ; 1,4-Naphthoquinone; oxonol V; 1,5-Bis(3-phenyl-5-oxoisoxazol-4-yl) pentamethine oxonol; TEMPO; 2,2,6,6-tetramethyl-piperidine-1-oxyl; Tm(DOTP); 5; −; thulium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphate); ΔΨ; transmembrane difference of electric potential23; Na-NMR; Complex I; Ion transport; NADH dehydrogenase; Proton; Sodium
Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments
by Denis V. Yanykin; Andrei A. Khorobrykh; Sergey A. Khorobrykh; Vyacheslav V. Klimov (pp. 516-523).
Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O2 photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O2 photoreduction on the acceptor side of PSII, there is light-induced O2 consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O2 uptake on the donor side of PSII is related to interaction of O2 with radicals produced by photooxidation of organic molecules. The study of flash-induced O2 uptake finds that removal of Mn from the WOC leads to O2 photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O2 evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O2 uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5–10μM, corresponding to 2–4 Mn per RC) of Mn2+, while at higher concentrations (>100μM) Mn2+ inhibits the O2 photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn2+) leads to both suppression of flash-induced O2 uptake and disappearance of the Mn-induced activation of the O2 photoconsumption. We assume that the light-induced O2 uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O2 or its reactive forms in the formation of the inorganic core of the WOC.
Keywords: Abbreviations; PSII; photosystem II; RC; reaction centre; WOC; water-oxidizing complex; Cyt; b; 559; cytochrome; b; 559; Pheo; pheophytin–the primary electron acceptor of PSII; P; 680; the primary electron donor of PSII; Q; A; the primary plastoquinone electron acceptor of PSII; Q; B; the secondary plastoquinone electron acceptor of PSII; TyrZ; redox active tyrosine residue of D1 protein; Diuron; 3-3,4-dichlorophenyl-1,1-dimethylurea; DPC; diphenylcarbazid; DCBQ; 2,6-dichloro-p-benzoquinone; ΔF; photoinduced changes of chlorophyll fluorescence yield of PSIIPhotosystem II; Oxygen photoconsumption; Manganese; Reactive oxygen species
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