FTIR detection of water reactions in the oxygen-evolving centre of photosystem II

Flash-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the water-oxidizing reactions in the oxygen-evolving centre of photosystem II. Reactions of water molecules were directly monitored by detecting the OH stretching bands of weakly H-bonded OH of water in the 3700-3500 cm(-1) region in FTIR difference spectra during S-state cycling. In the S1-->S2 transition, a band shift from 3588 to 3617 cm(-1) was observed, indicative of a weakened H-bond. Decoupling experiments using D2O:H2O (1:1) showed that this OH arose from a water molecule with an asymmetric H-bonding structure and this asymmetry became more significant upon S2 formation. In the S2-->S3, S3-->S0 and S0-->S1 transitions, negative bands were observed at 3634, 3621 and 3612 cm(-1), respectively, representing formation of a strong H-bond or a proton release reaction. In addition, using complex spectral features in the carboxylate stretching region (1600-1300 cm-(1)) as 'fingerprints' of individual S-state transitions, pH dependency of the transition efficiencies and the effect of dehydration were examined to obtain the information of proton release and water insertion steps in the S-state cycle. Low-pH inhibition of the S2-->S3, S3-->S0 and S0-->S1 transitions was consistent with a view that protons are released in the three transitions other than S1-->S2, while relatively high susceptibility to dehydration in the S2-->S3 and S3-->S0 transitions suggested the insertion of substrate water into the system during these transitions. Thus, a possible mechanism of water oxidation to explain the FTIR data is proposed.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

Below‐ground organic matter accumulation along a boreal forest fertility gradient relates to guild interaction within fungal communities

Plant–soil interactions link ecosystem fertility and organic matter accumulation below ground. Soil microorganisms play a central role as mediators of these interactions, but mechanistic understanding is still largely lacking. Correlative data from a coniferous forest ecosystem support the hypothesis that interactions between fungal guilds play a central role in regulating organic matter accumulation in relation to fertility. With increasing ecosystem fertility, the proportion of saprotrophic basidiomycetes increased in deeper organic layers, at the expense of ectomycorrhizal fungal species. Saprotrophs correlated positively with the activity of oxidative enzymes, which in turn favoured organic matter turnover and nitrogen recycling to plants. Combined, our findings are consistent with a fungus‐mediated feedback loop, which results in a negative correlation between ecosystem fertility and below‐ground carbon storage. These findings call for a shift in focus from plant litter traits to fungal traits in explaining organic matter dynamics and ecosystem fertility in boreal forests.     

Chemical Partitioning of Fe,Mn, Zn and Cr in Mountain Soils of the Iberian and Pyrenean Ranges (NE Spain)

The bioavailability of heavy metals, and thus their possible harmful impact on ecosystems and humans, depends on metal partitioning. This study describes the chemical partitioning of iron, manganese, zinc and chromium, because the potential effect of soil contamination is better assessed through the knowledge of the forms in which a given metal is present rather than the use of its total concentration. In four soil types representative of mountain soils in the Iberian and Pyrenean ranges in Aragón, a total of 16 selected sites have been sampled and five subsamples were taken in each site to create a composite sample. The 4 heavy metals have been extracted by the sequential extraction procedure of Tessier et al. (1979) Tessier, A., Campbell, P. G.C. and Bisson, N. 1979. Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem., 51: 844–850. [CROSSREF][Crossref], [Web of Science ®] [Google Scholar] and analyzed by emission atomic spectrometry of solid state (ICP OES). Little Fe, Mn and Zn (< 1, 2.4–35.9, < 1–24.5 mg/kg, respectively) were retrieved from the exchangeable phase, a readily available phase for biogeochemistry cycles in the ecosystems. Chromium was not detected in the potentially bioavailable forms. The largest contents of Fe, Zn and Cr were retrieved from the residual phase with mean values of 21100, 72 and 60 mg/kg, and maximum values of 35700, 279 and 271 mg/kg, respectively. Mn was mainly bound to oxide phases with mean and maximum values of 236 and 887 mg/kg, respectively. For all metals, the highest contents are found in Leptosols on igneous rocks due to the rich and diverse mineral composition of their parent materials. The knowledge of the chemical partitioning of Fe, Mn, Zn and Cr provides information to identify the soils in which their mobile forms may be transferred to the soil-water-plant system. This information is of interest for the management of fragile mountain soils to avoid the environmental risk of cycling these metals in the environment.     

Action of extracellular divalent cations on native alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors

The effects of divalent cations on Ca2+-impermeable containing (GluR2 subunit) MPA receptors of hippocampal pyramidal neurones isolated from rat brain was studied using patch-clamping. Ca2+, Mg2+, Mn2+, Co2+, Ni2+ and Zn2+ inhibited currents induced by kainate and glutamate. Inhibition was fast, reversible and voltage independent. The rank order of activities was Ni2+ > Zn2+ > Co2+ > Ca2+ > Mn2+ > Mg2+. Cyclothiazide (0.1 mm) significantly reduced inhibition by divalent cations and 6, 7 dinitroquinoxaline-2.3-dione (DNQX). However, high concentrations of Ni2+ and DNQX inhibited AMPA receptors even in the presence of cyclothiazide. The inhibitory effect of divalent cations as well as DNQX was counteracted by an increase in agonist concentration. In the presence of divalent cations the EC50 values of kainate and glutamate were increased, but the maximal response was not changed. An increase in agonist concentration induced a parallel shift in the concentration-inhibition curve for a divalent cation. These data suggest a competitive-like type of inhibition. However, an increase in agonist concentration reduced the inhibitory action of Ni2+ less than that of DNQX. This gave evidence against direct competition between divalent cations and AMPA receptor agonists. A 'complex-competition' hypothesis was proposed to explain the inhibitory action of divalent cations; it is suggested that divalent cations form ion-agonist complexes, which compete with free agonist for agonist-binding sites on AMPA receptors.     

A compact RNA tertiary structure contains a buried backbone-K+ complex

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.     

Evidence of two-step deprotonation of D-mannitol in aqueous solution

Deprotonation of D-mannitol was studied in aqueous basic solutions by means of potentiometry and (13)C NMR spectroscopy. Two-step dissociation in the pH range from 12 to 13.8 was shown, and successive dissociation constants K(a1) and K(a2) were determined. In a solution with ionic strength I = 1.0 M (NaOH + NaNO(3)) pK(a1) = 13.1 +/- 0.1 and pK(a2) = 13.8 +/- 0.2. With increasing ionic strength from 0.75 to 3.0 M, both pK(a1) and pK(a2) values decrease. Deprotonation-induced chemical shifts in pH-variable (13)C NMR spectra show that the OH-groups next to internal carbon atoms C-3 and C-4 dissociate to a greater extent compared to OH-groups next to external carbon atoms C-1 and C-6.     

Purification and some properties of superoxide dismutase from<Emphasis Type="Italic"> Deinococcus radiophilus</Emphasis>, the UV-resistant bacterium

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg^–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H₂O₂. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian     

Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors

The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri. We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT-PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (CapLC-ESI-QTOF-MS)-based NA analysis procedure and consistently found higher NA levels in roots of A. halleri. Expression of a NA synthase in Zn2+-hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri. Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species.     

Improvements in the determination of manganese peroxidase activity

The existing method of determining the activity of manganese peroxidase (MnP), produced by Phanerochaete chrysosporium, was improved. 2,6-Dimethoxyphenol at 80 mM was used as a substrate and, after the decolorization of the reaction mixture, H₂O₂ was added and the initial reaction rate was used to determine MnP activity.