Biospectroscopy最新文献

An experiment was proposed and accomplished that was based on the hypothesis of the dissociation of the luciferase–luciferin complex in photoexcitation. A pump–probe experiment was performed with the use of picosecond laser pulses and was based on the effect of quenching of enzyme tryptophan fluorescence caused by luciferin binding. A photoinduced increase of the tryptophan fluorescence intensity was detected. Experimental results were interpreted on the basis of the assumptions on photoinduced dissociation of the luciferin–luciferase complex and Forster energy transfer from tryptophan to luciferin. Under the assumption on the photoinduced dissociation and stationary quenching of tryptophan fluorescence the rate of propagation of the conformational changes in the protein caused by the complex dissociation was estimated to be >20 m/s. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 378–384, 1999

The secondary structure of four kinds of calmodulins (CaMs; i.e., Brassica campestris pollen CaM, bovine brain CaM, earthworm calcium binding protein, and earthworm new calcium binding protein) in thin films are determined by the FTIR resolution enhanced technique and curve fitting. The variation in the secondary structure of CaM upon its binding with Ca²⁺, Eu³⁺, and Tb³⁺, the assay of phosphodiesterase enzyme, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are also investigated. The effect of lanthanide ions on the conformation of CaM are described. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 371–377, 1999

Difference infrared spectra are reported for human serum transferrin in D₂O as a function of iron binding or increased acidity. Spectral features detected as iron is bound at high pH include difference bands that are indicative of reduced solvent exposure and binding site ligation. More extensive spectral alterations, some of which indicate titration of carboxylic acid groups, are induced in the apo protein by lowering the pH in a manner consistent with that entailed in endocytosis. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 325–327, 1999

A spectroscopic study of glutathione (GSH) and glutathione disulfide (GSSG) has been performed using Fourier-transformed infrared absorption and Raman scattering in order to pinpoint the sites of complexation of these two species with water and particularly with H₂O₂. Molecules of GSH and GSSG were studied in KBr pellets, and in aqueous solutions of H₂O, D₂O, and H₂O with H₂O₂ (1 mol L⁻¹) to characterize the specific influence of the solvent molecules. A time-resolved Raman study was performed for GSH/H₂O₂ in aqueous solution at 1 : 1 molar ratio in order to observe the formation of GSSG and to discuss the mechanism of this redox reaction. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 328–337, 1999

Resonance Raman spectroscopy and quantum chemical calculations were used to investigate the molecular origin of the large redshift assumed by the electronic absorption spectrum of astaxanthin in α-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. Resonance Raman spectra of α-crustacyanin reconstituted with specifically ¹³C-labeled astaxanthins at the positions 15, 15,15′, 14,14′, 13,13′, 12,12′, or 20,20′ were recorded. This approach enabled us to obtain information about the effect of the ligand–protein interactions on the geometry of the astaxanthin chromophore in the ground electronic state. The magnitude of the downshifts of the CC stretching modes for each labeled compound indicate that the main perturbation on the central part of the polyene chain is not homogeneous. In addition, changes in the 1250–1400 cm⁻¹ spectral range indicate that the geometry of the astaxanthin polyene chain is moderately changed upon binding to the protein. Semiempirical quantum chemical modeling studies (Austin method 1) show that the geometry change cannot be solely responsible for the bathochromic shift from 480 to 632 nm of protein-bound astaxanthin. The calculations are consistent with a polarization mechanism that involves the protonation or another interaction with a positive ionic species of comparable magnitude with both ketofunctionalities of the astaxanthin-chromophore and support the changes observed in the resonance Raman and visible absorption spectra. The results are in good agreement with the conclusions that were drawn on the basis of a study of the charge densities in the chromophore in α-crustacyanin by solid-state NMR spectroscopy. From the results the dramatic bathochromic shift can be explained not only from a change in the ground electronic state conformation but also from an interaction in the excited electronic state that significantly decreases the energy of the π-antibonding CO orbitals and the HOMO–LUMO gap. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 358–370, 1999

In this work, we have examined, using Fourier-transform Raman (FT-R) spectroscopy, the bacteriochlorophyll a (BChl a) binding sites in light-harvesting (LH) antennae from different species of the Proteobacteria that exhibit unusal absorption properties. While the LH1 complexes from Erythromicrobium (E.) ramosum (RC-B871) and Rhodospirillum centenum (B875) present classic FT-R spectra in the carbonyl high-frequency region, we show that in the blue-shifted LH1 complex, absorbing at 856 nm, from Roseococcus thiosulfatophilus, as well as in the B798–832 LH2 from E. ramosum, or in the B830 complex from the obligate phototrophic bacterium Chromatium purpuratum, some H-bonds between the acetyl carbonyl of the BChl a and the surrounding protein are missing. The molecular mechanisms responsible for the unusual absorption of these complexes are thus similar to those responsible for tuning of the absorption of the LH2 complexes between 850 and 820 nm. Furthermore, our results suggest that the binding pocket of the monomeric BChl in the LH2 from E. ramosum is different from that of Rps. acidphila or Rb. sphaeroides. The FT-R spectra of Chromatium purpuratum indicate that, in contrast with every LH2 complex previously studied by FT-R spectroscopy, no free-from-interaction keto groupings exist in this complex. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 338–345, 1999

Q_y-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPh_L) is replaced by a bacteriochlorophyll (BChl) molecule, designated β. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg⁺² ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg⁺² ion. The salient observations are as follows: (1) The β cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for β in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C₉-keto group of β in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C₉-keto of β is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of β and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of β in the protein is different in the two β-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 346–357, 1999

The ba₃ cytochrome oxidase from Thermus thermophilus was studied by resonance Raman spectroscopy. The component spectra of both heme groups were determined by using different excitation wavelengths. In the ferric state the heme a₃ group reveals resonance Raman marker bands characteristic for two high spin species with the heme iron in an in-plane and an out-of-plane configuration that reflects a coordination equilibrium. This equilibrium obviously results from protonation of one of the axial ligands that is ascribed to a hydroxide. Coordination by its protonated form, a water molecule, may be too weak to keep the heme iron in the porphyrin plane. The corresponding Fe-OH₂ stretching mode was attributed to a weak H/D-sensitive band at 464 cm⁻¹. The coordination equilibrium not only depends on the pH but is also affected by the buffer, the salt concentration, and the binding of the natural redox partner cytochrome c₅₅₂. These changes of the coordination equilibrium are attributed to the perturbation of the hydrogen bonding network at the catalytic center that is connected to the protein surface via a relay of hydrogen bonds. Environmental changes at the catalytic site are sensitively reflected by the formyl stretching of heme a₃. The unique structural properties of the ba₃ oxidase may be related to the unusual proton pump efficiency and heme a₃ redox potential. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: S53–S63, 1999

A series of oligopeptides containing aromatic and basic residues were synthesized and their interactions with double-stranded nucleic acids studied by proton and phosphorus NMR, viscometry, and DNA melting temperature (T_m). The oligopeptides prepared contain two aromatic amino acids (phenylalanine or p-nitrophenylalanine) as well as one or two lysyl residues. The nucleic acids studied were calf thymus DNA, poly(dA-dT)₂, poly(dA) · poly(dT), poly(dG-dC)₂, poly(dG) · poly(dC), and d(ATGCAT)₂. The results obtained show stacking of both aromatic residues of the oligopeptides with the nucleic acids. Higher upfield shifts of the aromatic amino acid residues were always observed with alternating nucleic acids and were higher with poly(dA-dT)₂ in all cases. Evidence for two types of complexes of Lys-Phe-Gly-Gly-p-NO₂Phe-LysNH₂ with DNA was obtained by NMR, one attributed to a purely electrostatic complex and another involving stacking interactions. Studies with d(ATGCAT)₂ indicate that the aromatic residues of the oligopeptides were stacked with the terminal AT base pairs preferentially binding at the ends of the hexanucleotide. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 313–322, 1999

The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4°C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b₅, a known bis-histidine complex. Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: S42–S52, 1999