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Etude par échange isotopique du radical tyrosyle en solution et dans la catalase bovine

Abstract : During the dispropornation of hydrogen peroxide yielding water and oxygen by catalase, a heme enzyme, radical intermediates of different nature are formed. Firstly, one species called Compound I appears, which is composed of one oxoferryl moiety coupled to a π-cation porphyrin radical. It has been shown by EPR measurements that for BLC (Beef Liver Catalase), one tyrosyl radical appears after few milliseconds, but the localization and the exact role of this radical remains obscure.
In our laboratory, we have developed a methodology based on tritium labelling to characterize the sites of hydroxyl radical attacks (created by water radiolysis) on each natural amino acid. This method was further used to label peptides, proteins and protein complex. When HO• reacts with the amino acid side chain, mainly by H-abstraction, it forms a carbon-centered radical. We used tritiated phenyl phosphinic sodium salt as a tritium atom donor to scavenge the produced radical. This irreversible label can then be easily analysed.
We have used this labelled compound to understand the sites of the radical production within the catalase by adding the substance when the radical has already formed. EPR measurements were done to monitor the decrease of the tyrosyl radical in the presence of the phosphinic agent. Preliminary results were rather encouraging as the radical seemed to be quenched by the added compound. When the labelling reaction was performed on the BLC, radioactivity was detected within the enzyme. Unfortunately, no specific labelling was detected when the enzyme was digested into peptides by a protease. As the initial compound was inefficient in that case, we decided to check if others structures would be able to transfer one tritium atom to the tyrosyl radical. After synthesis of such compounds, we have tested their reaction with the BLC tyrosyl radical by EPR spectroscopy, to check the decrease of the tyrosyl radical signal. Some of them showed an improved reactivity towards the tyrosyl radical than the initial compound.
In parallel, we have decided to investigate the chemistry of these compounds on a simpler system. We decided to produce the tyrosyl radical in solution via the reaction of tyrosine with hydroxyl radicals, generated by water radiolysis (γ rays). Once the radicals on tyrosine are produced, they were scavenged by a deuterium atom, provided by a 2H-donor. Tthe deuterium incorporation inside tyrosine was measured precisely by mass spectrometry as well as the regioselectivity by 2H NMR. During the course of this study, we also shed light on a so far unknown process, i.e. the self repair of tyrosyl radical. We then focused our attention on this phenomenon and proposed a mechanism to explain our observations.
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Sophie Oppilliart. Etude par échange isotopique du radical tyrosyle en solution et dans la catalase bovine. Chimie. Université Paris Sud - Paris XI, 2007. Français. ⟨tel-00361211⟩

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