The molecular changes of phytochrome during red --> far-red and reverse photoreactions have been monitored by static infrared difference spectroscopy using the recombinant 65 kDa N-terminal fragment assembled with a chromophore chemically modified at ring D or with a chromophore isotopically labeled with (18)O at the carbonyl group of ring A. This allows the identification of the C=O stretching vibrations of rings D and A. We exclude the formation of an iminoether in Pfr. The positions of both these modes show that the chromophore always remains protonated. The upshift of the C=O stretch of ring D in the first photoproducts is explained by a twisted methine bridge connecting rings C and D. The changes in the vibrational pattern during the red --> far-red conversion show that the backreaction is not just the reversal of the forward reaction. The infrared difference spectra of the fragment deviate very little from those of the full-length protein. The differences which are related to the lack of the C-terminal half of the protein constituting the signaling domain are possibly important for the understanding of the signaling mechanism.

The origin of the organic layer covering colloidal biogenic elemental selenium nanoparticles (BioSeNPs) is not known, particularly in the case when they are synthesized by complex microbial communities. This study investigated the presence of extracellular polymeric substances (EPS) on BioSeNPs. The role of EPS in capping the extracellularly available BioSeNPs was also examined. Fourier transform infrared (FT-IR) spectroscopy and colorimetric measurements confirmed the presence of functional groups characteristic of proteins and carbohydrates on the BioSeNPs, suggesting the presence of EPS. Chemical synthesis of elemental selenium nanoparticles in the presence of EPS, extracted from selenite fed anaerobic granular sludge, yielded stable colloidal spherical selenium nanoparticles. Furthermore, extracted EPS, BioSeNPs, and chemically synthesized EPS-capped selenium nanoparticles had similar surface properties, as shown by ζ-potential versus pH profiles and isoelectric point measurements. This study shows that the EPS of anaerobic granular sludge form the organic layer present on the BioSeNPs synthesized by these granules. The EPS also govern the surface charge of these BioSeNPs, thereby contributing to their colloidal properties, hence affecting their fate in the environment and the efficiency of bioremediation technologies.

The photocycle of 124 kDa phytochrome A from Avena sativa was studied by Fourier-transform infrared spectroscopy at low temperatures. Difference spectra between the parent state Pr and the intermediates of the Pr-->Pfr pathway, i.e. lumi-R, meta-Ra, and meta-Rc, and between Pfr and the intermediates of the Pfr-->Pr pathway, lumi-F and meta-F, were obtained in 1H2O and 2H2O for the first time. Each spectrum shows characteristic spectral features which allow a clear distinction between the different intermediates. A general feature is that greater changes occur with increasing temperature, i.e. at the later steps of the photoreactions. Nevertheless, the changes in the spectral regions of the protein (amide I and amide II) were found to be surprisingly small, excluding larger conformational changes of the protein. All spectra of the intermediates are characterized by a strong negative band around 1700 cm-1. This band is tentatively assigned to the C = O stretch of ring D of the chromophore. Since it is not observed in the difference spectra between the parent states, it is concluded that ring D is located in a similar molecular environment in Pr and Pfr. In the photoproducts lumi-R and lumi-F, this band undergoes an upshift to 1720 cm-1. The high frequencies suggest that the chromophore is protonated in these intermediates as well as in Pr and Pfr.

The speciation of uranium(VI) in micromolar aqueous solutions at ambient atmosphere was studied by attenuated total reflection Fourier-transform infrared (ATR FT-IR) spectroscopy and by speciation modeling applying the updated NEA thermodynamic database. It can be shown that reliable infrared spectra of micromolar U(VI) solutions are obtained abolishing the restrictions of previous spectroscopic investigations to millimolar concentrations and, consequently, to the acidic pH range. A significant change of the U(VI) speciation can be derived from the spectral alterations of the absorption band representing the antisymmetric stretching mode (nu3) of the UO2(2+) ion observed upon lowering the U(VI) concentration from the milli- to the micromolar range at a constant pH 4 value. The acquisition of spectra of diluted U(VI) solutions allows the increase of the pH up to 8.5 without the risk of formation of colloidal or solid phases. The infrared spectra are compared to the results of the computed speciation patterns. Although a complete interpretation of the spectra can not be given at this state of knowledge, the spectral data strongly suggest the presence of monomeric U(VI) hydroxo species already showing up at a pH value > or = 2.5 and dominating the speciation at pH 3. This is in contradiction to the predicted speciation where the fully hydrated UO2(2+) is expected to represent the main species at pH values below 4. At ambient pH, a more complex speciation is suggested compared to the results of the computational modeling technique. The predicted dominance of the UO2(CO3)3(4-) complex at pH > or = 8 was not confirmed by the infrared data. However, the infrared spectra indicate the formation of hydroxo complexes obviously containing carbonate ligands.

We employed density functional theory (DFT) calculations, and ultraviolet-visible (UV-vis), extended X-ray absorption fine-structure (EXAFS), and attenuated total reflection Fourier-transform infrared (IR) spectroscopy analyzed with iterative transformation factor analysis (ITFA) to determine the structures and the pH-speciation of aqueous acetate (ac) and succinate (suc) U(VI) complexes. In the acetate system, all spectroscopies confirm the thermodynamically predicted pH-speciation by Ahrland (1951), with the hydrated uranyl ion and a 1:1, a 1:2 and a 1:3 U(VI)-ac complex. In the succinate system, we identified a new 1:3 U(VI)-suc complex, in addition to the previously known 1:1 and 1:2 U(VI)-suc complexes, and determined the pH-speciation for all complexes. The IR spectra show absorption bands of the antisymmetric stretching mode of the uranyl mojety (υ3(UO2)) at 949, 939, 924 cm(-1) and at 950, 938, 925 cm(-1) for the 1:1, 1:2 and 1:3 U(VI)-ac and U(VI)-suc complexes, respectively. IR absorption bands at 1535 and 1534 cm(-1) and at 1465 and 1462 cm(-1) are assigned to the antisymmetric υ3,as(COO) and symmetric υ3,s(COO) stretching mode of bidentately coordinated carboxylic groups in the U(VI)-ac and U(VI)-suc complexes. The assignment of the three IR bands (υ3(UO2), υ3,as(COO), υ3,s(COO)) and the stoichiometry of the complexes is supported by DFT calculations. The UV-vis spectra of the equivalent U(VI)-ac and U(VI)-suc complexes are similar suggesting common structural features. Consistent with IR spectroscopy and DFT calculations, EXAFS showed a bidentate coordination of the carboxylic groups to the equatorial plane of the uranyl moiety for all uranyl ligand complexes except for the newly detected 1:3 U(VI)-suc complex, where two carboxylic groups coordinate bidentately and one carboxylic group coordinates monodentately. All 1:1 and 1:2 complexes have a U-Owater distance of ∼2.36 Å, which is shorter than the U-Owater distance of ∼2.40 Å of the hydrated uranyl ion. For all complexes the U-Ocarboxyl distance of the bidentately coordinated carboxylic group is ∼2.47 Å, while the monodentately coordinated carboxylic group of the 1:3 U(VI)-suc complex has a U-Ocarboxyl distance of ∼2.36 Å, that is, similar to the short U-Owater distance in the 1:1 and 1:2 complexes.

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The interconvertible photoreactions of recombinant phytochrome from Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below -100 degrees C for the transformation of red-absorbing form of phytochrome (Pr)-->far-red-absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at -140 degrees C (lumi-R) was observed. Two intermediates could be stabilized below -40 degrees C and between -40 and -20 degrees C, and were denoted as meta-Ra and meta-Rc, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The FT-IR difference spectra of the late Pr-->Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-Ra were observed. A large band around 1700 cm-1 in the difference spectra between the intermediates and Pr which is tentatively assigned to the C19=O group of the prosthetic group indicates the Z,E isomerization around the C15=C16-methine bridge of the chromophore during the formation of meta-Ra. In the difference spectra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of 2H2O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm-1 in all 2H2O spectra suggests an assignment to an N-2H bending vibration of the chromophore.

Citrate complexes are the dominant binding form of trivalent actinides and lanthanides in human urine at pH < 6. Hence, an accurate prediction of the speciation of these elements in the presence of citrate is crucial for the understanding of their impact on the metabolism of the human organism and the corresponding health risks. We studied the complexation of Cm(III) and Eu(III), as representatives of trivalent actinides and lanthanides, respectively, in aqueous citrate solution over a wide pH range using time-resolved laser-induced fluorescence spectroscopy. Four distinct citrate complexes were identified and their stability constants were determined, which are MHCit(0), M(HCitH)HCit(2-), M(HCit)(2)(3-), and M(Cit)(2)(5-) (M = Cm, Eu). Additionally, there were also indications for the formation of MCit(-) complexes. Structural details on the EuHCit(0) and EuCit(-) complexes were obtained with FT-IR spectroscopy in combination with density functional theory calculations. IR spectroscopic evidence for the deprotonation of the hydroxyl group of the citrate ion in the EuCit(-) complex is presented, which also revealed that the complexation of the Eu(3+) ion takes place not only through the carboxylate groups, like in EuHCit(0), but additionally via the hydroxylate group. In both EuHCit(0) and EuCit(-) the carboxylate binding mode is mono-dentate. Under a very low metal : citrate ratio that is typical for human body fluids, the Cm(III) and Eu(III) speciation was found to be strongly pH-dependent. The Cm(III) and Eu(III) citrate complexes dominant in human urine at pH < 6 were identified to be Cm(HCitH)HCit(2-) and a mixture of Eu(HCitH)HCit(2-) and EuHCit(0). The results specify our previous in vitro study using natural human urine samples (Heller et al., Chem. Res. Toxicol., 2011, 24, 193-203).