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Etude structurale de métalloprotéines à centres [2Fe-2S].
Cas d'une ferrédoxine et d'une dioxygénase impliquée dans la biodégradation des
hydrocarbures aromatiques.
Cristallographie des protéines à très haute énergie.
Méthodes de phasage d'une protéine modele à 55 keV.

Abstract : Metalloproteins containing Fe-S clusters play an important role in nature as they are
involved in essential physiological functions including photosynthesis, respiration, and
nitrogen fixation. In this thesis, a [2Fe-2S] ferredoxin involved in Fe-S cluster biogenesis, and
a bacterial dioxygenase playing a critical role in aromatic hydrocarbon biodegradation were
subjected to structural analysis by synchrotron X-ray crystallography. The structure of a
ferredoxin from the photosynthetic bacterium Rhodobacter capsulatus was solved in both its
oxidized and reduced states. Subtle structural changes were observed upon reduction,
especially in the vicinity of the [2Fe-2S] cluster. These changes are discussed in comparison
with those described for ferredoxins with similar structures but different functions.
A more complex metalloprotein, belonging to a large family of bacterial dioxygenases,
was studied for its ability to oxidize polycyclic aromatic hydrocarbons (PAHs). This multicomponent
enzyme, isolated from a PAH-degrading Sphingomonas strain, consists of a
NAD(P)H-oxidoreductase, a [2Fe-2S] ferredoxin, and a terminal oxygenase. The terminal
oxygenase component, called PhnI, consists of six subunits assembled into an ?3?3 hexamer,
and contains one Rieske-type [2Fe-2S] cluster and one Fe(II) ion per ? subunit, which were
identified by their characteristic EPR signature. The enzyme showed an exceptionally broad
substrate specificity, as it could hydroxylate a wide range of PAHs made of two to five fused
rings, including the carcinogens, benz[a]anthracene and benzo[a]pyrene. With naphthalene as
substrate, steady-state kinetics showed that the enzyme had a low apparent Km (0.92 WM) and
a specificity constant of 2.0 WM-1. s-1. The Phn1 protein was crystallized and its threedimensional
structure was determined at 1.85 A resolution. In spite of moderate sequence
similarity with homologous dioxygenases, the 3D polypeptide fold was found to be very
similar, most of the differences being observed near the substrate binding pocket.
Many protein crystals, especially those of Fe-S proteins, have been shown to undergo
X-ray radiation damage, leading to artifacts in protein structure determinations. As an attempt
to solve the problem, ultra-high energy X-rays (55 keV; 0.22 A), which are only slightly
absorbed by proteins, were used for the first time to determine the 3D structure of a model
protein, lysozyme. Beamline specificities as well as optimum energy were determined.
Potential applications for structural biology are discussed.
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https://tel.archives-ouvertes.fr/tel-00170921
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Submitted on : Monday, September 10, 2007 - 9:16:08 PM
Last modification on : Friday, February 26, 2021 - 12:38:02 PM
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Jean Jakoncic. Etude structurale de métalloprotéines à centres [2Fe-2S].
Cas d'une ferrédoxine et d'une dioxygénase impliquée dans la biodégradation des
hydrocarbures aromatiques.
Cristallographie des protéines à très haute énergie.
Méthodes de phasage d'une protéine modele à 55 keV.. Autre [q-bio.OT]. Université Joseph-Fourier - Grenoble I, 2007. Français. ⟨tel-00170921⟩

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