Thioesters spectroscopy

The effect of antioxidants such as hindered phenohcs, secondary amine, and thioester on the radiation cross-linking efficiency of LDPE has been reported [260]. Amount of cross-linking at a given dose decreases with aU the antioxidants, the thioester being the most effective. IR absorption spectroscopy has been used to demonstrate dose-rate dependence of trani -vinylene unsaturation in irradiated Marlex 50 PE [261]. When the irradiated polymer is stored in vacuum a decrease is observed in trani-vinylene absorbance over a period of several weeks. After high dose-rate irradiation the decay is preceded by an initial increase. These phenomena have been ascribed to the reaction of trapped radicals. 

The Photoactive Yellow Protein (PYP) is the blue-light photoreceptor that presumably mediates negative phototaxis of the purple bacterium Halorhodospira halophila [1]. Its chromophore is the deprotonated trans-p-coumaric acid covalently linked, via a thioester bond, to the unique cystein residue of the protein. Like for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of the PYP photocycle, but the reaction path that leads to the formation of the cis isomer is not clear yet (for review see [2]). From time-resolved spectroscopy measurements on native PYP in solution, it came out that the excited-state deactivation involves a series of fast events on the subpicosecond and picosecond timescales correlated to the chromophore reconfiguration [3-7]. On the other hand, chromophore H-bonding to the nearest amino acids was shown to play a key role in the trans excited state decay kinetics [3,8]. In an attempt to evaluate further the role of the mesoscopic environment in the photophysics of PYP, we made a comparative study of the native and denatured PYP. The excited-state relaxation path and kinetics were monitored by subpicosecond time-resolved absorption and gain spectroscopy. 

The Photoactive Yellow Protein (PYP) is thought to be the photoreceptor responsible for the negative phototaxis of the bacterium Halorhodospira halophila [1]. Its chromophore, the deprotonated 4-hydroxycinnamic (or p-coumaric) acid, is covalently linked to the side chain of the Cys69 residue by a thioester bond. Trans-cis photoisomerization of the chromophore was proved to occur during the early steps of the PYP photocycle. Nevertheless, the reaction pathway leading to the cis isomer is still discussed (for a review, see ref. [2]). Time-resolved spectroscopy showed that it involves subpicosecond and picosecond components [3-7], some of which could correspond to a flipping motion of the chromophore carbonyl group [8,9]. 

In order to better understand the early photophysics of PYP, we have carried out a comparative study of three model chromophores, the deprotonated frans-p-coumaric acid (pCA2 ) and its amide (pCM ) and phenyl thioester (pCT) analogues, in aqueous solution (see structures in Fig. 1). The excited-state relaxation dynamics was followed by subpicosecond transient absorption and gain spectroscopy. 

Mossbauer spectroscopy has proved useful in investigating the series of complexes [FeBr(NO)2L] in which L is a sulphur donor ligand. A distinctly different Mossbauer isomer shift for the o-aminothiophenol (atp) compounds, compared with the monodentate thioester complexes (Table II), is evidence for a change from 4- to 5-coordination in the former (97). 

In the formate esters, the free energy difference decreases from esters to thioesters and amides for the methyl and tert-butyl groups, and E-methyl formate is only observed in very small amounts (<0.3%) at low temperature (190 K), as shown by 13C NMR spectroscopy [3], The energy is significantly lower for the bulky tert-butyl moiety relative to the methyl group and this seems to be a function of steric hindrance since ethyl formate and iso-propyl formate have intermediary values [4,5]. In the tert-butyl formate, a strong electronic repulsion accounts for the relatively high proportion of E isomer (10%). 

The paramagnetism of all oxidation states except Cr(VI) and the highly reactive nature of Cr(VI) esters with biomolecules, make NMR spectroscopy of limited use for the characterization of Cr complexes however, multinuclear NMR spectroscopy has been used to characterize Cr(VI) thioester complexes (98, 99) and NMR spectroscopy has enabled Cr(VI)-esters with the oxalate ligand to be characterized (100). While these are challenging experiments and often require the use of labeled ligands and low temperatures to enable spectra to be recorded before the intermediates decompose, it is a valuable technique for some applications. 

Since the NMR resonances of the carbonyl of thioesters are shifted (A 20 to 30 ppm), NMR spectroscopy should allow the direct monitoring of the formation and decay of a thioacyl intermediate. Using = O] N-benzoylimidazole (5 = 168.7 ppm), we were able to observe directly a thioacyl intermediate at 8 = 195.9 ppm in the presence of papain under the cryoenzymological conditions of -t C in 25 percent aqueous dimethyl sulfoxide. Moreover, the thioacyl species is clearly a productive intermediate since the decrease in its signal intensity was accompanied by an increase in the product resonances and by release of free enzyme (half-life, 96 minutes) determined by titration of its thiol group. The line width of the resonance at 195.9 ppm was 25 Hz [5,6]. 

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