Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (Y_D) in PsbD, the D2 subunit of plant photosystem II, were 50.8?±?3.5?Å, 54.9?±?4.0?Å, 57.8?±?4.9?Å, and 58.4?±?14.1?Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of Y_D relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed Y_D coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.     

Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two-hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP-bound form of Rho1p. This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature-sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p.     

Function of chlorophyll d in reaction centers of photosystems I and II of the oxygenic photosynthesis of Acaryochloris marina

Reaction center chlorophylls (Chls) in photosystems II and I were studied in the isolated thylakoid membranes of a cyanobacterium, Acaryochloris marina, which contains Chls d and a as the major and minor pigments, respectively. The membranes contained PS I and II complexes at a 1.8:1 molar ratio on the basis of the spin densities on the tyrosine D radical and the photo-oxidized PS I primary donor (P740+). In the presence of ferricyanide, laser excitation induced bleach at 725 nm that recovered with time constants of 25 micros and 1.2 ms. The signal, designated P725, was suppressed by PS II inhibitors DCMU and hydroxylamine. The P725 spectrum was tentatively assigned to the absorption changes of the special pair Chl d, the accessory Chl d, and the acceptor pheophytin a in PS II. The addition of ascorbate induced the additional signal with a slow decay time constant of 4.5 ms. This signal showed a broad bleach at 740 nm and shift-type absorption changes at around 707 and 685 nm, which were assigned to the absorption changes of PS I special pair of Chl d (P740), the accessory Chl d, and the primary acceptor Chl a (A0), respectively. Mechanisms and the evolution of the Chl-d based reaction centers using far-red light are discussed together with the amino acid sequences of PS II D1 and D2 proteins.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S(0) and S(2) states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D(2)O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S(0) and S(2) state multiline disappeared after 3 h of D(2)O incubation in the S(0) and S(1) states, respectively. The signal with 4.0 MHz hyperfine constants in S(0) state multiline disappeared after 3 h of D(2)O incubation in the S(0) state, while the similar signal in S(2) state multiline disappeared only after 24 h of D(2)O incubation in the S(1) state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 A, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae,isolated from coral reef seawater off Ishigaki Island,Japan

Two phylogenetically distinct Vibrionaceae strains C4II189^T and C4V358^T isolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189^T (=DSM 101554^T = JCM 31629^T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358^T (=DSM 101555^T = JCM 31630^T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus.     

Unique binding site for Mn2+ ion responsible for reducing an oxidized YZ tyrosine in manganese-depleted photosystem II membranes.

Binding of Mn2+ to manganese-depleted photosystem II and electron donation from the bound Mn2+ to an oxidized YZ tyrosine were studied under the same equilibrium conditions. Mn2+ associated with the depleted membranes in a nonsaturating manner when added alone, but only one Mn2+ ion per photosystem II (PS II) was bound to the membranes in the presence of other divalent cations including Ca2+ and Mg2+. Mn2+-dependent electron donation to photosystem II studied by monitoring the decay kinetics of chlorophyll fluorescence and the electron paramagnetic resonance (EPR) signal of an oxidized YZ tyrosine (YZ+) after a single-turnover flash indicated that the binding of only one Mn2+ ion to the manganese-depleted PS II is sufficient for the complete reduction of YZ+ induced by flash excitation. The results indicate that the manganese-depleted membranes have only one unique binding site, which has higher affinity and higher specificity for Mn2+ compared with Mg2+ and Ca2+, and that Mn2+ bound to this unique site can deliver an electron to YZ+ with high efficiency. The dissociation constant for Mn2+ of this site largely depended on pH, suggesting that a single amino acid residue with a pKa value around neutral pH is implicated in the binding of Mn2+. The results are discussed in relation to the photoactivation mechanism that forms the active manganese cluster.     

Detection of Peramivir and Laninamivir,New Anti-Influenza Drugs,in Sewage Effluent and River Waters in Japan

This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs—oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN—in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.     

ESR signal of the iron-sulfur center F(X) and its function in the homodimeric reaction center of Heliobacterium modesticaldum

Electron transfer in the membranes and the type I reaction center (RC) core protein complex isolated from Heliobacterium modesticaldum was studied by optical and ESR spectroscopy. The RC is a homodimer of PshA proteins. In the isolated membranes, illumination at 14 K led to accumulation of a stable ESR signal of the reduced iron-sulfur center F(B)(-) in the presence of dithiothreitol, and an additional 20 min illumination at 230 K induced the spin-interacting F(A)(-)/F(B)(-) signal at 14 K. During illumination at 5 K in the presence of dithionite, we detected a new transient signal with the following values: g(z)= 2.040, g(y)= 1.911, and g(x)= 1.896. The signal decayed rapidly with a 10 ms time constant after the flash excitation at 5 K and was attributed to the F(X)(-)-type center, although the signal shape was more symmetrical than that of F(X)(-) in photosystem I. In the purified RC core protein, laser excitation induced the absorption change of a special pair, P800. The flash-induced P800(+) signal recovered with a fast 2-5 ms time constant below 150 K, suggesting charge recombination with F(X)(-). Partial destruction of the RC core protein complex by a brief exposure to air increased the level of the P800(+)A(0)(-) state that gave a lifetime (t(1/2)) of 100 ns at 77 K. The reactions of F(X) and quinone were discussed on the basis of the three-dimensional structural model of RC that predicts the conserved F(X)-binding site and the quinone-binding site, which is more hydrophilic than that in the photosystem I RC.