The uptake characteristics of Prussian-blue nanoparticles for rare metal ions for recycling of precious metals from nuclear and electronic wastes.

We have examined the uptake mechanisms of platinum-group-metals (PGMs) and molybdenum (Mo) ions into Prussian blue nanoparticles (PBNPs) in a nitric acid solution for 24-h sorption test, using inductively coupled plasma atomic emission spectroscopy, powder XRD, and UV-Vis-NIR spectroscopy in combination with first-principles calculations, and revealed that the Ru4+ and Pd2+ ions are incorporated into PBNPs by substitution with Fe3+ and Fe2+ ions of the PB framework, respectively, whereas the Rh3+ ion is incorporated into PBNPs by substitution mainly with Fe3+ and minorly with Fe2+ ion, and Mo6+ ion is incorporated into PBNPs by substitution with both Fe2+ and Fe3+ ions, with maintaining the crystal structure before and after the sorption test. Assuming that the amount of Fe elusion is equal to that of PGMs/Mo substitution, the substitution efficiency is estimated to be 39.0% for Ru, 47.8% for Rh, 87% for Pd, and 17.1% for Mo6+. This implies that 0.13 g of Ru, 0.16 g of Rh, 0.30 g of Pd, and 0.107 g of Mo can be recovered by using 1 g PBNPs with a chemical form of KFe(III)[Fe(II)(CN)6].

The uptake mechanism of palladium ions into Prussian-blue nanoparticles in a nitric acid solution toward application for the recycling of precious metals from electronic and nuclear wastes.

We have investigated the uptake mechanism of palladium (Pd: one of the most important elements in industry used as a catalyst) ions into Prussian-blue nanoparticles (PBNPs) in a nitric acid solution via high-resolution electron transmission microscopy, inductively coupled plasma atomic emission spectroscopy, powder X-ray diffraction, and ultraviolet-visible-near infrared spectroscopy in combination with first principles calculations. Comparison of the structural and electronic properties of PBNPs between before and after a 24 h sorption test reveals that the Pd2+ ions incorporated into PBNPs by the substitution of Fe2+ ions of the PB framework while maintaining the crystal structure, and the substitution efficiency is estimated to be 87% per PB unit cell. This implies that 0.30 g of Pd can be recovered by using 1 g of PB having the chemical formula KFe(iii)[Fe(ii)(CN)6]. The present finding suggests that PB (or its analogues) can be applied to recycle noble and rare metals from electronic and nuclear wastes.

Hydrothermal-treatment desorption of cesium from clay minerals: The roles of organic acids and implications for soil decontamination.

The adsorption and desorption of cesium (Cs) on clays of contaminated soil in a rhizosphere zone can be greatly affected by various biogeochemical processes, the timespans of which are usually months to years. Herein, we present several representative scenarios of the binding of Cs on diverse sites of vermiculitized biotite by controlled Cs adsorption to particles of different sizes. We investigated whether and how the fixed Cs in the different scenarios is desorbed by ambient and hydrothermal treatments with several low-molecular-weight organic acids (LMWOAs). The results showed that the sorbed Cs was discriminatively retained in the un-collapsed, partially collapsed, and thoroughly collapsed structures of vermiculites. The desorption of the sorbed Cs by hydrothermal LMWOAs extractions was easily realized in the un-collapsed structure, but was limited or minimal in the partially collapsed and thoroughly collapsed structures. The Cs desorption varied in accord with the LMWOA species applied and increased with the acid concentration, temperature, and number of treating cycles. The analysis of Cs-desorbed specimens confirmed their partial destruction and interlayer expansion, suggesting that the underlying mechanism of Cs removal by LMWOAs involves not only acid dissolution and complexation but also the accelerated weathering of clays within a short time under hydrothermal conditions. Our findings contribute novel insights into the mobility, bioavailability, and fate of Cs in contaminated soils and its removal from these soils for environmental restorations.

Regulation of Photocycle Kinetics of Photoactive Yellow Protein by Modulating Flexibility of the ß-Turn.

The role of the significant flexibility of the ß-turn in photoactive yellow protein (PYP) due to Gly115 was studied. G115A and G115P mutations were observed to accelerate the photocycle and shift the equilibrium between the late photocycle intermediate (pB) and its precursor (pR) toward pR. Thermodynamic analysis of dark-state recovery from pB demonstrated that the transition state (pB⧧) has a negative change in transition heat capacity, suggesting that an exposed hydrophobic surface of pB is buried in pB⧧. Fourier transform infrared spectroscopy showed that the structural ensemble of pB is populated by the compact structure in G115P. Taken together, the rigid structure induced by mutation of Gly115 facilitates its transition to pB⧧, which adopts a substantially more compact structure as opposed to the ensemble-averaged structure of pB. The photocycle kinetics of PYP may be fine-tuned by modulating the flexibility of the 115 loop to activate an appropriate number of transducer proteins.

Interaction between N-terminal loop and beta-scaffold of photoactive yellow protein.

During the photoreaction cycle of photoactive yellow protein (PYP), a physiologically active intermediate (PYP(M)) is formed as a consequence of global protein conformational change. Previous studies have demonstrated that the photocycle of PYP is regulated by the N-terminal loop region, which is located across the central beta-sheet from the p-coumaric acid chromophore. In this paper, the hydrophobic interaction between N-terminal loop and beta-sheet was studied by characterizing PYP mutants of the hydrophobic residues. The rate constants and structural changes of the photocycle of L15A and L23A possibly participating in such an interaction were more similar to wild-type than F6A, showing that the CH/pi interaction between Phe6 and Lys123 is the most essential as reported previously. To better understand the interactions between N-terminal tail and beta-sheet of PYP, Phe6 and Phe121 were replaced by Cys and linked by a disulfide bond. Since the photocycle kinetics, structural change and thermal stability of F6C/F121C were similar to F6A, the CH/pi interaction between Phe6 and Lys123 is not substitutable. It is likely that the detachment of position 6 from position 123 substantially alters the nature of PYP.

Low-temperature spectroscopy of Met100Ala mutant of photoactive yellow protein.

The trans-to-cis photoisomerization of the p-coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis-to-trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV-visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100A(BL)), while the corresponding intermediate of wild type (WT; PYP(BL)) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100A(B') and M100A(L)) were substantially less than those from WT. While the near-UV intermediate (PYP(M)) is not formed from WT in glycerol samples at low temperature, M100A(M) was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis-chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 A), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle.

Diverse roles of glycine residues conserved in photoactive yellow proteins.

The role of glycine residues was studied by alanine-scanning mutagenesis using photoactive yellow protein, a structural prototype of PER ARNT SIM domain proteins, as a template. Mutation of glycine located close to the end of beta-strands with dihedral angles disallowed for alanine (Gly-37, Gly-59, Gly-86, and Gly-115) induces destabilization of the protein structure. On the other hand, substitution for Gly-77 and Gly-82, incorporated into the fifth alpha-helix, slows the photocycle by 15-20 times, suggesting that these residues regulate the light-induced structural switch between dark-state structure and signaling-state structure. Most importantly, a significant amount of G29A is in the bleached state and showed a 1000-fold slower photocycle. As O(epsilon2) of the carboxylic acid of Glu-46 is close enough for contact with C(alpha) of Gly-29, alanine mutation perturbs this packing. Fourier transform infrared spectroscopy demonstrated that the C=O(epsilon2) stretching mode of Glu-46 is 6 cm(-1) upshifted in G29A, suggesting that C(alpha) of Gly-29 acts as a proton donor for the C(alpha)-H...O(epsilon2) hydrogen bond with Glu-46, which stabilizes the dark-state structure. During the photocycle, Glu-46 becomes negatively charged by donating a proton to the chromophore, resulting in breakage of this hydrophobic packing and consequent conformational change of the protein.

Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering.

It is widely accepted that PYP undergoes global structural changes during the formation of the biologically active intermediate PYP(M). High-angle solution x-ray scattering experiments were performed using PYP variants that lacked the N-terminal 6-, 15-, or 23-amino-acid residues (T6, T15, and T23, respectively) to clarify these structural changes. The scattering profile of the dark state of intact PYP exhibited two broad peaks in the high-angle region (0.3 A(-1) < Q < 0.8 A(-1)). The intensities and positions of the peaks were systematically changed as a result of the N-terminal truncations. These observations and the agreement between the observed scattering profiles and the calculated profiles based on the crystal structure confirm that the high-angle scattering profiles were caused by intramolecular interference and that the structure of the chromophore-binding domain was not affected by the N-terminal truncations. The profiles of the PYP(M) intermediates of the N-terminally truncated PYP variants were significantly different from the profiles of the dark states of these proteins, indicating that substantial conformational rearrangements occur within the chromophore-binding domain during the formation of PYP(M). By use of molecular fluctuation analysis, structural models of the chromophore-binding region of PYP(M) were constructed to reproduce the observed profile of T23. The structure obtained by averaging 51 potential models revealed the displacement of the loop connecting beta4 and beta5, and the deformation of the alpha4 helix. High-angle x-ray scattering with molecular fluctuation simulation allows us to derive the structural properties of the transient state of a protein in solution.

Array of aromatic amino acid side chains located near the chromophore of photoactive yellow protein.

The role of the array of aromatic amino acid side chains located close to the chromophore binding loop of photoactive yellow protein (PYP) was studied using the alanine-substitution mutagenesis. Phe92, Tyr94, Phe96 and Tyr98 were replaced with alanine (F92A, Y94A, F96A and Y98A, respectively), then these mutants were characterized by UV-visible absorption spectra, circular dichroism (CD) spectra, thermal stability and photocycle kinetics. Absorption maxima of F92A, Y94A, F96A and Y98A were 444, 442, 439 and 447 nm, respectively, different to wild type (WT) at 446 nm. Far-UV CD spectra of mutants other than F92A were different from WT, indicating that Tyr94, Phe96 and Tyr98 maintain the native secondary structure of PYP. Mid-point temperatures of thermal denaturation of F92A, Y94A and F96A, estimated by the CD signal at 222 nm, were 5-10 degrees C lower than WT. Time constants of the photocycle estimated by flash-induced absorbance change were 0.36 s for WT and 1.4 s for Y98A, however, 100, 30 and 3000 times slower than WT for F92A, Y94A and F96A, respectively. Tyr98 is located in the loop region, whereas Phe92, Tyr94 and Phe96 are incorporated in the beta4 strand, showing that aromatic amino acid residues in the beta-sheet regulate the absorption spectrum, thermal stability and photocycle of PYP. Aromatic rings of Phe92, Tyr94 and Phe96 lie nearly perpendicular to the aromatic ring of Phe75 or chromophore. Possible weak hydrogen bonds between the aromatic ring hydrogen and pi-electrons of these residues are discussed.

Stilbene analogs in Hula-twist photoisomerization.

Photoisomerization of several cis- or Z-stilbene analogs and two E-analogs in low temperature organic glasses was examined. From a mechanistic view-point, the compounds can be divided into three types: (i) those giving identical Hula-twist (HT) and one-bond-flip (OBF) products, (ii) those giving a single HT product that is different (hence distinguishable) from the OBF product and (iii) those showing two distinct HT processes but only one OBF process. Examples for all three types of analogs are provided emphasizing the most informative Type-II (stilbene analogs with identical but unsymmetrically substituted phenyl rings), including linear as well as conformationally constrained compounds. Conditions necessary for establishing HT and OBF processes are defined. Proper choice and design of model systems are essential for establishing or eliminating HT mechanism(s) of isomerization.

A single CH/pi weak hydrogen bond governs stability and the photocycle of the photoactive yellow protein.

Importance of the CH/pi interaction on the structure and function of the photoactive yellow protein (PYP) was substantiated. Focusing on the phenyl ring of Phe6 adjacent to the alkyl chain of Lys123, the mutants for these amino acid residues were characterized. The results demonstrated that the mutants lacking the pi-electron at position 6 or the alkyl chain at position 123 show substantial malfunction. This is a clear example that single CH/pi weak interaction plays a crucial role in the normal action of the protein.

Conformational changes of PYP monitored by diffusion coefficient: effect of N-terminal alpha-helices.

Conformational changes in the light illuminated intermediate (pB) of photoactive yellow protein (PYP) were studied from a viewpoint of the diffusion coefficient (D) change of several N-truncated PYPs, which lacked the N-terminal 6, 15, or 23 amino acid residues (T6, T15, and T23, respectively). For intact PYP (i-PYP), D of pB (D(pB)) was approximately 11% lower than that (D(pG)) of the ground state (pG) species. The difference in D (D(pG) - D(pB)) decreased upon cleavage of the N-terminal region in the order of i-PYP>T6>T15>T23. This trend clearly showed that conformational change in the N-terminal group is the main reason for the slower diffusion of pB. This slower diffusion was interpreted in terms of the unfolding of the two alpha-helices in the N-terminal region, increasing the intermolecular interactions due to hydrogen bonding with water molecules. The increase in friction per one residue by the unfolding of the alpha-helix was estimated to be 0.3 x 10(-12) kg/s. The conformational change in the N-terminal group upon photoillumination is discussed.

pH-dependent equilibrium between long lived near-UV intermediates of photoactive yellow protein.

The long lived intermediate (signaling state) of photoactive yellow protein (PYP(M)), which is formed in the photocycle, was characterized at various pHs. PYP(M) at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP(M)(acid)) and alkaline form (PYP(M)(alkali)) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK(a) was 6.4. Content of alpha- and/or beta-structure of PYP(M)(acid) was significantly greater than PYP(M)(alkali) as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of beta-structure in the difference Fourier transform infrared spectra for formation of PYP(M)(acid) was smaller than that of PYP(M)(alkali). The vibrational mode at 1747 cm(-1) of protonated Glu-46 was found as a small band for PYP(M)(acid) but not for PYP(M)(alkali), suggesting that Glu-46 remains partially protonated in PYP(M)(acid), whereas it is fully deprotonated in PYP(M)(alkali). Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP(M)(acid) was 15.7 Angstroms, whereas for PYP(M)(alkali) it was 16.2 Angstroms. These results indicate that PYP(M)(acid) assumes a more ordered and compact structure than PYP(M)(alkali). Binding of citrate shifts this equilibrium toward PYP(M)(alkali). UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP(M)(acid), indicating that the mutation shifts this equilibrium toward PYP(M)(acid). Alterations in the nature of PYP(M) by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP(M)(acid) and PYP(M)(alkali).

Direct observation of the pH-dependent equilibrium between L-like and M intermediates of photoactive yellow protein.

Equilibrium between the photoproducts of photoactive yellow protein (PYP), present in a millisecond time scale, was studied. The near-UV intermediate of PYP (PYPM) was red-shifted by alkalization due to the deprotonation of the chromophore (pKa=10.2). In addition, a small amount of red-shifted intermediate coexisted with PYPM. Its spectral shape in the visible region agreed with that of PYPL, the precursor of PYPM. The fraction of PYPL-like product was maximal at pH 10. It decays with a rate constant identical to that of PYPM. These results indicate that PYPL-like product is in pH-dependent equilibrium with PYPM and deprotonated PYPM.

Role of an N-terminal loop in the secondary structural change of photoactive yellow protein.

Photoactive yellow protein (PYP) is photoconverted to its putative active form (PYP(M)) with global conformational change(s). The changes in the secondary structure were studied by far-UV circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy using PYP, which lacks N-terminal 6, 15, or 23 amino acid residues (T6, T15, and T23, respectively). Irradiation of truncated PYPs induced the loss of the CD signal, where the maximal difference was located at 222 nm. The reduction of the CD signal was significantly larger than the calculated CD of the N-terminal helices, indicating that it is mainly accounted for by the unfolding and/or structural change of the helices located outside the N-terminal region. The difference FTIR spectra between dark and photosteady states recorded using the solution samples demonstrated that large absorbance changes in the amide mode of the beta-sheet were reduced and downshifted by truncation. The structural change of the beta-sheet is therefore closely correlated with the N-terminal loop. NaCl decelerates the decay of intact PYP(M) and T6(M) at low concentrations (<500 mM) but accelerates decay at high concentrations (>1000 mM). For T15(M) and T23(M), NaCl accelerates their decay at >100 mM but never decelerates their decay, suggesting that the electrostatic interaction, which plays an important role for the recovery of PYP from PYP(M), is lost by removing positions 7-15. The electrostatic interaction between this region and the beta-scaffold is likely to promote the conformational change of PYP(M) for recovery of PYP.

Light-induced global conformational change of photoactive yellow protein in solution.

The light-induced global conformational change of photoactive yellow protein was directly observed by small-angle X-ray scattering (SAXS). The N-terminal 6, 15, or 23 amino acid residues were enzymatically truncated (T6, T15, or T23, respectively), and their near-UV intermediates were accumulated under continuous illumination for SAXS measurements. The Kratky plot demonstrated that illumination induced partial loss of globularity. The change in globularity was marked in T6 but very small in T15 and T23, suggesting that structural change in positions 7-15 mainly reduces the globularity. The radius of gyration (R(g)) estimated by Guinier plot was increased by 1.1 A for T6 and 0.7 A for T15 and T23 upon illumination. As T23 lacks most of the N-terminal loop, structural change in the main part composed of the PAS core, helical connector, and beta-scaffold caused an increase of R(g) by 0.7 A. The structural change of positions 7-15 caused an additional increase by 0.4 A. The decrease of R(g) upon truncation of positions 7-15 for dark state was 0.3 A, while that for the intermediate was 0.7 A, suggesting that this region moves outward on formation of the intermediate. These results indicate that a light-induced structural change of PYP takes place in the main part and N-terminal 15 amino acid residues. The former induces only dimensional increase, but the latter results in additional change in shape.