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Etude des relations structure-fonctions de l´hydrogénase à fer de Clostridium acetobutylicum et de ses partenaires d'oxydo-réduction

Abstract : The biological production of hydrogen, a "clean" energy carrier, has recently aroused a great interest. The most efficient reported micro-organism for hydrogen production from hexose is the anaerobic bacterium Clostridium acetobutylicum with a chemostat production of 2.4 l H2 l-1 h-1, catalyzed by the HydA [FeFe]-hydrogenase. In order to understand and improve C. acetobutylicum hydrogen potentialities, we have attempted to characterize the structure-function relationships of HydA with its redox partners. By homology with the [FeFe]-hydrogenase of Clostridium pasteurianum, the [4Fe- 4S] FS4C and [2Fe-2S] FS2 clusters located on the protein surface were predicted to be involved in the inter-molecular electron transfer between HydA and its redox partners. Our goal was to determine the implication of the FS4C and FS2 clusters in this transfer. To do so, mutagenesis by amino acid substitutions and domain deletions was performed on FS4C and FS2. In order to stabilize native and modified hydrogenases, the purification protocol was improved. Native hydrogenase was successfully stabilized, while the persisting instability of modified hydrogenase was an obstacle of their catalytic characterization. Decreased activity of modified hydrogenases was due to a loss of active site functionality and to a lower iron content than the theoretical value. The physiological redox partners of HydA, ferredoxine CAC0303 and flavodoxin, were purified. The complete catalytic profile of HydA and its kinetic parameter with different redox partners for hydrogen uptake and hydrogen production were determined. The optimized purification protocol led to a significant increase of both hydrogen uptake and hydrogen production activities. We confirmed that the in vitro hydrogen uptake was more favourable than the hydrogen production. A very high kcat was determined with the artificial substrate methyl viologen for the hydrogen uptake activity. This result might indicate that methyl viologen could be prone to interact more or less directly with the active site, shunting the intra-molecular electron transfer chain. High catalytic efficiencies for both H2 uptake and H2 production activities were observed with either artificial (except for reduced methyl viologen) and physiological redox partners. This result reflects the high potential of HydA for hydrogen-related activities which is conserved with three different electron carriers. Hence, in iron-limited growth conditions, the in vivo substitution of ferredoxin by flavodoxin might not be a limitation for in vivo hydrogenase activity.
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Contributor : Marie Demuez <>
Submitted on : Monday, November 23, 2009 - 4:20:32 PM
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  • HAL Id : tel-00435141, version 1


Marie Demuez. Etude des relations structure-fonctions de l´hydrogénase à fer de Clostridium acetobutylicum et de ses partenaires d'oxydo-réduction. Biochimie [q-bio.BM]. INSA de Toulouse, 2007. Français. ⟨tel-00435141⟩



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