IR Difference Spectra Research Articles

Characterization of the primary electron donor of photosystem I, P700, by electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy

Abstract The redox reactions of P700, the primary electron donor of photosystem I (PS I), have been analyzed by electrochemistry and Fourier transform infrared (FTIR) spectroscopy in two different preparations from the cyanobacterium Synechocystis PCC 6803 yielding monomeric and trimeric PS I complexes, respectively. Reversible and quantitative oxidation and rereduction of P700 has been achieved at surface-modified electrodes in an optically and infrared transparent thin-layer electrochemical (OTTLE) cell. The midpoint potential ( E m = +260 ± 10 mV vs. Ag/AgCl/3 M KCl) determined from these titrations, either by monitoring the electrochemically-induced oxidation/reduction of the primary electron donor P700 at 700 nm or by monitoring the amplitude of the flash-induced absorbance change at 707 nm as a function of the applied potential, agree between monomeric and trimeric PS I, demonstrating that protein-protein contact has very little impact on the redox properties of P700. Using electrochemical oxidation and rereduction, the vibrational IR difference spectra P700 ·+ − P700 could be generated. In the 1800−1200 cm −1 spectral region, highly structured IR difference spectra were obtained. The IR difference bands titrate in unison and are fully reversible upon application of a reducing potential. The midpoint potential measured by titration of the absorbance difference of two prominent bands, at 1714 and 1696 cm −1 ( E m = +260 ± 10 mV) corresponds to the values determined by titration of the electronic transition of P700. In contrast to previously obtained FTIR difference spectra for light-induced charge separation (E. Nabedryk, M. Leonhard, W. Mantele and J. Breton, Biochemistry, 29 (1990) 3242; G. MacDonald, K.A. Bixby and B.A. Barry, Proc. Natl. Acad. Sci. USA, 90 (1993) 11024) which include contributions from the primary electron donor and the acceptor side, the electrochemical generated FTIR difference spectra exclusively reflect the molecular changes at P700 and its protein site upon removal of an electron. Nevertheless, they largely agree with the difference spectra of the charge-separated state. In the 1800-1680 cm −1 range, the IR difference signals are attributed to the keto and ester C=O modes of P700. Between 1700 and 1680 cm −1 , shifts of bands and changes of band intensities for photosystem I particles suspended in 2 H 2 O reveal contributions of amide I modes. Further effects of 2 H 2 O/ 1 H 2 O exchange are observed between 1600 and 1500 cm −1 , and around 1400 cm −1 . Between 1750 cm −1 and approximately 3000 cm −1 , no FTIR difference signals were detected. In particular, no broad but strong absorbance changes were detected around 2600 cm −1 , where a low-energy electronic transition, characteristic for the dimeric nature of P and P + , was detected for P865 in bacterial reaction centers (J. Breton, E. Nabedryk and W. Parson, Biochemistry, 31 (1992) 7503). This indicates that the positive charge on P700 ·+ is rather localized on one of the chlorophylls.

Femtosecond time-resolved infrared laser study of the J− K transition of bacteriorhodopsin

The J− K transition of the bacteriorhodopsin photocycle was monitored by sub-picosecond time-resolved infrared spectroscopy. IR difference spectra in the region between 1670 and 1600 cm −1 were taken at 1.5 and 9 ps, respectively, after photoexcitation of BR 570 at 540 nm. Spectral shifts of the bands at 1607 and 1661 cm −1 reflect the chromophoric conformational changes during the J to K transition. Kinetics, taken at 1640 and 1607 cm −1 show rise times determined by the dephasing times of the vibrational modes. The partial decrease of the bleach signal at 1640 cm −1 is interpreted as a recovery of the vibrationally cooled BR 570 electronic ground state and provides a new method to measure the photocycle quantum yield. The development of the bleach at 1661 cm −1 occurred faster than 500 fs, suggesting an almost instantaneous protein response to the electronic excitation.

Infrared spectroscopic detection of light-induced change in chloride-arginine interaction in halorhodopsin

A light-induced transient change in the ionic interaction between chloride and arginine in the transmembrane anion pump halorhodopsin (hR) is detected with infrared absorption spectroscopy. In the IR difference spectrum of hR and one of its photoproducts (hL), only a few bands have frequencies that depend on the particular halide ion (Cl-, Br-, or I-) present. Three of the halide-sensitive negative difference bands (at 1695, 1610, and 1170 cm-1) correspond in frequency to arginine C-N vibrations and undergo anion-dependent shifts that match those seen in ethylguanidinium halide model compounds. These shifts reflect the different strengths of the ionic interactions formed with the various halides. We conclude that a halide-arginine ion pair is present in the hR state; this interaction appears to be disrupted by photoconversion to hL.

Proton transfer processes in the hydrogen-bonded structure of the active centre of serine proteases — an FT—IR study

FT—IR difference spectra of trypsin + inhibitor minus anhydrotrypsin + inhibitor complexes were used to study the proton transfer processes in the active centres of serine proteases. The modification of Ser 195 (chymotrypsinogen numbering) is clearly indicated by the difference spectra of trypsin minus anhydrotrypsin. This modification changes the environment of the Asp 102His 57 arrangement. At pH 3 the proton in the OH ⋯ N ⇌ O − ⋯ H +N hydrogen bond is shifted to Asp 102 with anhydrotrypsin whereas with trypsin a protonated His 57 residue is observed, indicating that in this case the equilibrium is completely shifted to the right hand side. Direct information on the catalytic mechanism is obtained by the bands of serine, histidine and aspartic acid in the difference spectra of trypsin + soybean trypsin inhibitor (SBTI) minus anhydrotrypsin + SBTI, as well as those of trypsin + bovine pancreatic trypsin inhibitor (BPTI) minus anhydrotrypsin + BPTI. In the case of the trypsin + SBTI complex a tetrahedral intermediate (TI)-like state is not formed at pH 3. It is, however, partially formed at pH 7. With the trypsin + BPTI complex the TI-like state is already partially formed at pH 3. At pH 7 the ES ⇌ TI equilibrium is shifted in favour of the right hand side. In both cases His 57 is protonated in this state. His 57 remains protonated in the TI due to the strong interaction of the Asp 102His 57 hydrogen bond with its environment since this hydrogen bond shows large proton polarizability. Thus, the serine −OH is not activated by a shift of the positive charge in a charge relay system. Instead an acid catalysis by the histidine residue is the true mechanism. It is discussed that the protonated histidine in the TI is favourable for the next step of the mechanism.

Protein response to photodissociation of CO from carbonmonoxymyoglobin probed by time-resolved infrared spectroscopy of the amide I band.

Protein conformational changes coupled to the ligation reactions of carbon monoxide in myoglobin (Mb) are detected by time-resolved infrared spectroscopy. An apparatus based on a tunable diode laser operating in the region of 1650 cm-1 is used to probe changes in the amide I absorption band of the protein in response to photodissociation and subsequent rebinding of CO. The time course of changes in the amide I band is shown to follow the recombination of photolyzed CO with Mb. A time-resolved difference spectrum in the amide I region is generated by tuning the diode laser probe source. The features in the IR difference spectrum are assigned to the motions of the polypeptide backbone associated with the global relaxation of the protein from the ligated to the deoxy conformation. A static difference spectrum generated by subtracting FTIR spectra of carbonmonoxy-Mb and deoxy-Mb is essentially identical to the transient spectrum, indicating that the protein relaxation is complete with the 100-ns time resolution of the experiment.

Protein conformational changes in tetraheme cytochromes detected by FTIR spectroelectrochemistry: Desulfovibrio desulfuricans Norway 4 and Desulfovibrio gigas cytochromes c3

The conformational change coupled to the redox processes of two tetraheme cytochromes c3 from bacteria of the genus Desulfovibrio have been studied by UV-vis and FTIR difference spectroscopy combined with protein electrochemistry. Two pairs of equivalent hemes were found in Desulfovibrio desulfuricans Norway 4 cytochrome c3 by UV-vis spectroelectrochemical redox titration in an optically transparent thin-layer electrochemical cell. In contrast to this, Desulfovibrio gigas cytochrome c3 showed a UV-vis difference spectrum for the highest potential heme different from that of the others. The redox titrations were monitored by FTIR difference spectroscopy using the same spectroelectrochemical cell. They show that in both cytochromes the overall redox process from the fully oxidized (III4) to the fully reduced oxidation state (II4), III4<==>II4, proceeds via an intermediate oxidation stage (III2II2) which is formed after the second electron uptake. The small amplitude of the difference signals in the reduced-minus-oxidized FTIR difference spectra obtained for the overall redox process in both Desulfovibrio cytochromes indicates a very small conformational change induced by the redox transition. Nevertheless, by application of potential steps from the fully oxidized or reduced form to the midwave potential (as obtained from the UV-vis redox titrations), the reduced-minus-oxidized IR difference spectra corresponding to the intermediate redox transitions (III4<==>III2II2 and III2II2<==>II4) were obtained, reflecting separately the contributions of the high- and low-potential heme pairs to the overall redox-induced conformational change. The overall redox process and both intermediate redox transitions were fully reversible. In the spectral region between 1500 and 1200 cm-1 the IR difference spectra of both cytochromes show several signals previously observed in the reduced-minus-oxidized IR difference spectra of spinach cytochrome b559 and iron-protoporphyrin IX-bis(imidazole) model compounds [Berthomieu, C., Boussac, A., Mäntele, W., Breton, J., & Nabedryk, E. (1992) Biochemistry 31, 11460-11471]. Moreover, Raman spectra of Desulfovibrio vulgaris cytochrome c3 and cytochrome b5 show signals attributed to Raman active heme skeletal modes at nearly the same positions [Kitagawa, T., Kyogoyu, Y., Izuka, T., Ikeda-Saito, M., & Yamanaka, T. (1975) J. Biochem. 78, 719-728], thus allowing their assignment to signals arising from heme vibrational modes. Comparatively strong IR difference signals at 1618 cm-1, which are tentatively assigned to phenylalanine residues, were found in D. desulfuricans cytochrome c3. In the spectra of D. gigas cytochrome c3, IR signals at 1614 cm-1 were detected only for the first redox transition (III4<==>III2II2).(ABSTRACT TRUNCATED AT 400 WORDS)

Electrochemically induced conformational changes in cytochrome c monitored by Fourier transform infrared difference spectroscopy: influence of temperature, pH, and electrode surfaces.

IR difference spectra between the oxidized and the reduced state of horse heart cytochrome c were obtained for different temperature and pH conditions at various surface-modified electrodes using an optically transparent thin-layer electrochemical cell. These difference spectra reflect changes in protein conformation, side-chain geometries, and protonation upon the redox transition. The IR difference spectra recorded in the 10-40 degrees C temperature range showed thermally induced changes mainly in the amide-I (1700-1600 cm-1) and in the amide-II (ca. 1550 cm-1) spectral regions. Although the position of most of the signals remains unshifted, large differences in their relative amplitude were observed, leading in some cases to the masking and/or the disappearance of some IR signals. In the range 6.8-9.8, increasing pH of the samples led to a decrease in the reduction rate and to spectral changes which closely resemble those obtained by increasing the temperature. Both the thermal and the pH dependence of the reduced-minus-oxidized IR difference spectra reflect the transition of ferricytochrome c from the native to the alkaline form. An analysis of the IR difference spectra shows that the redox transition at neutral pH involves mainly beta-turns and beta-sheet segments of the cytochrome c molecule. However, once the ferricytochrome c alkaline transition is performed, the redox process is coupled to conformational changes involving alpha-helical segments. The shifts in tyrosine vibrational modes observed in the difference spectra obtained at neutral and slightly alkaline pH at high temperatures suggest an intermediate state of the ferricytochrome c in which the heme crevice is more accessible to the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein dynamics in the bacteriorhodopsin photocycle: submillisecond Fourier transform infrared spectra of the L, M, and N photointermediates.

The usefulness of stroboscopic time-resolved Fourier transform IR spectroscopy for studying the dynamics of biological systems is demonstrated. By using this technique, we have obtained broadband IR absorbance difference spectra after photolysis of bacteriorhodospin with a time resolution of approximately 50 microseconds, spectral resolution of 4 cm-1, and a detection limit of delta A less than or equal to 10(-4). These capabilities permit observation of detailed structural changes in individual residues as bacteriorhodopsin passes through its L, M, and N intermediate states near physiological temperatures. When combined with band assignments based on isotope labeling and site-directed mutagenesis, the stroboscopic Fourier transform IR difference spectra show that on the time scale of the L intermediate, Asp-96 has an altered environment that may be accompanied by change in its protonation state. On the time scale of the L----M transition, this Asp-96 perturbation/deprotonation is largely reversed, and Asp-85 becomes protonated. During the M----N transition, Asp-85 appears to remain protonated but undergoes a change in its environment as evidenced by a shift of vC = O from 1761 to 1755 cm-1. The retention of a proton on Asp-85 in the N state indicates that the proton transferred from the Schiff base to this residue in the L----M step is not released to the extracellular medium during the same photocycle, but rather during a subsequent one. Also during the M----N transition, Asp-96 undergoes a deprotonation (possibly for the second time in a single photocycle). Bands in the amide I and amide II spectral regions in the M----N difference spectrum indicate the occurrence of a conformational change involving one or more peptide groups in the protein backbone.

An investigation of some aspects of the weathering of plasticized PVC

Two types of commercial plasticized opaque PVC films (0·16 mm thick), based on different kinds of suspension PVC have been investigated. Colourimetry, difference IR-spectra, MIR-IR spectra and elongation at break have been used for the assessment of changes during one year of weathering. Interesting results have been obtained with difference IR-spectra in the initial stages of exposure. Carbonyl absorption on the front surface layers of the specimens revealed non-monotonous development. During weathering, short polyenes accumulate to a smaller extent in the bulk of modified PVC based samples in comparison with the control. A relationship between discoloration and changes in double bond content on the exposed side is observed as well as autocatalytic changes in the later period. The behaviours of the two films tested and a discrepancy in the results obtained by different methods have been discussed.

A high-resolution electron microscopy investigation of TiO 2(B)-supported vanadium oxide catalysts

TiO 2(B) crystals were found to be isomorphic with those of its precursor, K 2Ti 4O 9. The former crystals had a large number of facetted voids, 3–30 nm, formed as a result of the removal of potassium and water in preceding hydrolysis and calcination steps, respectively. TiO 2(B)-supported vanadium oxide catalysts with loadings in the range 0.25−10 theoretical layers were prepared by impregnation of the support with an oxalic acid solution of NH 4VO 3 followed by calcination in air. HREM micrographs of catalysts with a low vanadium loading, recorded using a low electron-dose imaging technique, showed that the surfaces, in the initial stage, were without any anomalous surface structure. This observation may be due to a similar structure of the support and the deposited vanadia phase. At high vanadium loadings, both amorphous and crystalline particles were seen, in agreement with the features revealed by the use of IR spectroscopy. For catalysts with low vanadium loadings, the IR difference spectra showed the presence of tetrahedrally coordinated V 4+ and V 5+ species. In a fully converged electron beam, reduction of the support and the vanadia phases occured, resulting in the formation of small crystallites. The catalysts were used for the oxidation of toluene to benzaldehyde. However, contrary to what has been observed for the ammoxidation producing benzonitrile, no enhanced catalytic properties, in comparison with those of crystalline V 2O 5, were obtained using TiO 2(B) as support.

Spectroscopic investigation of ternary solutions: H 2OCH 3CNCu(ClO 4) 2

Over a large range of concentrations in the ternary solutions H 2OCH 3CHCu(ClO 4) 2, information about the various intermolecular interactions are extracted from the characteristic spectra of each component present: water (OH/D stretching vibration), CH 3CN (CN stretching vibration), Cu 2+ (electronic transition on the visible region) and (ClO 4) − ion (ClO stretching vibration). The formation of contact ion pairs seems to become important only at salt concentrations above 0.05 mol l −1. Using weighted IR difference spectra WDS it is possible to separate the spectrum of hydrate-water of the copper(II) ion from other H-bonded complexes present. The independent determination of the number of water and cyanomethane molecules interacting with the copper(II) ion is in agreement with a solvation number of 6. The composition of the solvation shell of the cation depends strongly on the composition of the solvent mixture. Qualitative agreement is found between these experimental results and theoretical considerations.

The Ability of the Proline Residue to Promote Successive-Intramolecular Hydrogen Bonds in Oligopeptides

Abstract In order to investigate the ability of the Pro residue to promote helical folding in oligopeptides, the IR spectroscopic conformational study of the oligo(Leu)s containing the Pro residue was performed in dichloromethane. Their IR difference spectra suggested the occurrence of successive-intramolecular hydrogen bonds just like incipient α-helical structures formed by one, two, three, and so forth i→i–4 hydrogenbonding patterns, alluding to the ability of the Pro residue to promote helical folding in the peptides. However, the intensities of hydrogen-bonded absorption bands in the amide A region of each peptide strongly suggested that conformations of the peptides investigated were also contributed by other intramolecularly hydrogen-bonded folded structures, which had lower absorption coefficients. The initiation mechanism of successive-intramolecular hydrogen bonding in the peptides is apparently attributed to the disturbance of the β-sheet formation by the rotation of the tertiary peptide bond plane (Leu–Pro), that is, the restriction of the values of the backbone dihedral angles φ and ψ of the Pro residue due to the low flexibility of the pyrrolidine ring.

IR spectral study of a-Si:H films prepared by H-doping evaporation method

The IR difference spectra of a-Si:H films prepared by H-doping evaporation (H.D.E.) method were compared with those by glow discharge(G.D.) method. It has been found that the bonding H content of H.D.E. sample is comparable to that of G.D. sample. The behaviour of absorption bonds of dehydrogenated samples seems to show that the Si-H bonds in H.D.E. sample are more stable than those in G.D. sample.