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Le recteur de l'EPF, Günther Dissertori, remet à Charlotte Bopp le prix Otto Jaag pour la protection des eaux 2023 (Photo: EPF, Giulia Marthaler)

Prix Otto Jaag pour la protection des eaux 2023 et médaille EPF pour Charlotte Bopp

21 novembre 2023 | Claudia Carle

Charlotte Bopp, chercheuse en sciences de l’environnement, est doublement récompensée pour sa thèse: elle a reçu le 18 novembre le prix Otto Jaag pour la protection des eaux lors de la Journée de l’EPF et recevra également la médaille EPF en janvier. Son travail fournit une importante contribution à la compréhension de la biodégradation des polluants organiques dans l’environnement.

Avec le prix Otto Jaag pour la protection des eaux, l’EPF Zurich récompense l’excellence des mémoires de master et des thèses de doctorat dans le domaine de la protection des eaux et des sciences aquatiques. Cette distinction a été remise à Charlotte Bopp lors de la Journée de l’EPF le 18 novembre pour sa thèse intitulée «The role of oxygen uncoupling by Rieske non-heme iron dioxygenases in the biodegradation of aromatic contaminants». La médaille EPF attribuée par l’EPF Zurich aux mémoires de master et aux thèses de doctorat d’excellence lui sera également décernée en janvier.

Oxydation inefficace des polluants

En tant que doctorante à l’Institut de recherche sur l’eau Eawag au département Chimie de l’environnement, Charlotte Bopp a étudié de près la biodégradation de polluants organiques difficilement biodégradables. Si de tels composés aromatiques, présents par exemple dans les pesticides, les médicaments ou les explosifs, parviennent dans le sol et les cours d’eaux, les micro-organismes peuvent les oxyder et les dégrader grâce à un groupe d’enzymes appelées «oxygénases de Rieske». Dans son travail, la chercheuse s’est concentrée sur le sous-groupe d’enzymes dégradant les explosifs et a cherché à déterminer leur niveau d’efficacité. Ses résultats ne donnent pas une bonne note aux enzymes. Plutôt que de transférer directement l’oxygène sur les polluants, les enzymes produisent d’abord une forme d’oxygène particulièrement réactive. La moitié de cet oxygène réactif seulement réagit ensuite effectivement avec les polluants, l’autre partie oxydant toutes sortes d’autres substances dans les micro-organismes, ce qui peut les désavantager, voire leur nuire.

Les nouvelles sont plus efficaces

Mais ce processus peut aussi présenter des avantages, comme l’a montré Charlotte Bopp: s’ils entrent en contact avec de nouveaux polluants pour la dégradation desquels leur spectre d’enzymes existant n’est pas approprié, les micro-organismes peuvent s’adapter. L’oxygène réactif provoque des mutations ponctuelles dans les enzymes, ce qui entraîne la modification de certains de leurs acides aminés et donc la création de nouvelles enzymes. Certaines d’entre elles travaillent même plus efficacement que les originales. Grâce à ce processus évolutif, les micro-organismes peuvent au bout d’un certain temps utiliser les nouveaux polluants.

«Le travail de recherche mené par Charlotte Bopp a permis de découvrir des liens inconnus jusqu’alors dans la biodégradation des polluants», déclare Thomas Hofstetter, responsable du département Chimie de l’environnement à l’Eawag et directeur de la thèse de la chercheuse. Jusqu’à présent, la capacité de dégradation des polluants était déduite de la quantité d’enzymes présentes dans l’environnement. «Les résultats de Charlotte Bopp montrent qu’il faut y regarder de plus près et prendre aussi en compte les différences d’efficacité des organismes et de leurs enzymes.»

La chercheuse se réjouit de la grande reconnaissance de son travail: «Nous avons osé regarder où ces enzymes semblent échouer.» Or, c’est précisément cette défaillance qui permet aux micro-organismes de faire face à long terme à de multiples polluants. «Nous avons pu résoudre cette énigme grâce à l’organisation interdisciplinaire de l’Eawag et à une équipe dont le travail commun a mérité ces distinctions», explique Charlotte Bopp. Depuis l’achèvement de sa thèse, elle travaille dans l’industrie au développement de processus d’ozonisation dans le traitement des eaux usées et de l’eau potable.
 

Photo de couverture: Le recteur de l'EPF, Günther Dissertori, remet à Charlotte Bopp le prix Otto Jaag pour la protection des eaux 2023 (Photo: EPF, Giulia Marthaler)
 

Publications originales

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         Hofstetter, T. B.' (103 chars)
      title => protected'Elucidating the role of O<sub>2</sub> uncoupling in the oxidative biodegrada
         tion of organic contaminants by Rieske non-heme iron dioxygenases' (141 chars)
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      startpage => protected'428' (3 chars)
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      categories => protected'non-heme ferrous iron oxygenases; Rieske oxygenases; biocatalysis; O2 uncoup
         ling; O2 activation; kinetic isotope effect; biodegradation' (135 chars)
      description => protected'Oxygenations of aromatic soil and water contaminants with molecular O<sub>2<
         /sub> catalyzed by Rieske dioxygenases are frequent initial steps of biodegr
         adation in natural and engineered environments. Many of these non-heme ferro
         us iron enzymes are known to be involved in contaminant metabolism, but the 
         understanding of enzyme-substrate interactions that lead to successful biode
         gradation is still elusive. Here, we studied the mechanisms of O<sub>2</sub>
          activation and substrate hydroxylation of two nitroarene dioxygenases to ev
         aluate enzyme- and substrate-specific factors that determine the efficiency 
         of oxygenated product formation. Experiments in enzyme assays of 2-nitrotolu
         ene dioxygenase (2NTDO) and nitrobenzene dioxygenase (NBDO) with methyl-, fl
         uoro-, chloro-, and hydroxy-substituted nitroaromatic substrates reveal that
          typically 20-100% of the enzyme's activity involves unproductive paths of O
         <sub>2</sub> activation with generation of reactive oxygen species through s
         o-called O<sub>2</sub> uncoupling. The <sup>18</sup>O and <sup>13</sup>C kin
         etic isotope effects of O<sub>2</sub> activation and nitroaromatic substrate
          hydroxylation, respectively, suggest that O<sub>2</sub> uncoupling occurs a
         fter generation of Fe<sup>III</sup>-(hydro)peroxo species in the catalytic c
         ycle. While 2NTDO hydroxylates <em>ortho</em>-substituted nitroaromatic subs
         trates more efficiently, NBDO favors <em>meta</em>-substituted, presumably d
         ue to distinct active site residues of the two enzymes. Our data implies, ho
         wever, that the O<sub>2</sub> uncoupling and hydroxylation activity cannot b
         e assessed from simple structure-reactivity relationships. By quantifying O<
         sub>2</sub> uncoupling by Rieske dioxygenases, our work provides a mechanist
         ic link between contaminant biodegradation, the generation of reactive oxyge
         n species, and possible adaptation strategies of microorganisms to the expos
         ure of new contaminants.' (1924 chars)
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      authors => protected'Pati,&nbsp;S.&nbsp;G.; Bopp,&nbsp;C.&nbsp;E.; Kohler,&nbsp;H.-P.&nbsp;E.; Ho
         fstetter,&nbsp;T.&nbsp;B.' (101 chars)
      title => protected'Substrate-specific coupling of O<sub>2</sub> activation to hydroxylations of
          aromatic compounds by rieske non-heme iron dioxygenases' (132 chars)
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      categories => protected'non-heme ferrous iron oxygenases; nitrobenzene dioxygenase; biocatalysis; O2
          uncoupling; isotope effects; xenobiotics' (117 chars)
      description => protected'Rieske dioxygenases catalyze the initial steps in the hydroxylation of aroma
         tic compounds and are critical for the metabolism of xenobiotic substances. 
         Because substrates do not bind to the mononuclear non-heme Fe<sup>II</sup> c
         enter, elementary steps leading to O<sub>2</sub> activation and substrate hy
         droxylation are difficult to delineate, thus making it challenging to ration
         alize divergent observations on enzyme mechanisms, reactivity, and substrate
          specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygena
         se capable of transforming nitroarenes to nitrite and substituted catechols,
          that unproductive O<sub>2</sub> activation with the release of the unreacte
         d substrate and reactive oxygen species represents an important path in the 
         catalytic cycle. Through correlation of O<sub>2</sub> uncoupling for a serie
         s of substituted nitroaromatic compounds with <sup>18</sup>O and <sup>13</su
         p>C kinetic isotope effects of dissolved O<sub>2</sub> and aromatic substrat
         es, respectively, we show that O<sub>2</sub> uncoupling occurs after the rat
         e-limiting formation of Fe<sup>III</sup>-(hydro)peroxo species from which su
         bstrates are hydroxylated. Substituent effects on the extent of O<sub>2</sub
         > uncoupling suggest that the positioning of the substrate in the active sit
         e rather than the susceptibility of the substrate for attack by electrophili
         c oxygen species is responsible for unproductive O<sub>2</sub> uncoupling. T
         he proposed catalytic cycle provides a mechanistic basis for assessing the v
         ery different efficiencies of substrate hydroxylation vs unproductive O<sub>
         2</sub> activation and generation of reactive oxygen species in reactions ca
         talyzed by Rieske dioxygenases.' (1703 chars)
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      doi => protected'10.1021/acscatal.2c00383' (24 chars)
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Elucidating the role of O2 uncoupling in the oxidative biodegradation of organic contaminants by Rieske non-heme iron dioxygenases

Oxygenations of aromatic soil and water contaminants with molecular O2 catalyzed by Rieske dioxygenases are frequent initial steps of biodegradation in natural and engineered environments. Many of these non-heme ferrous iron enzymes are known to be involved in contaminant metabolism, but the understanding of enzyme-substrate interactions that lead to successful biodegradation is still elusive. Here, we studied the mechanisms of O2 activation and substrate hydroxylation of two nitroarene dioxygenases to evaluate enzyme- and substrate-specific factors that determine the efficiency of oxygenated product formation. Experiments in enzyme assays of 2-nitrotoluene dioxygenase (2NTDO) and nitrobenzene dioxygenase (NBDO) with methyl-, fluoro-, chloro-, and hydroxy-substituted nitroaromatic substrates reveal that typically 20-100% of the enzyme's activity involves unproductive paths of O2 activation with generation of reactive oxygen species through so-called O2 uncoupling. The 18O and 13C kinetic isotope effects of O2 activation and nitroaromatic substrate hydroxylation, respectively, suggest that O2 uncoupling occurs after generation of FeIII-(hydro)peroxo species in the catalytic cycle. While 2NTDO hydroxylates ortho-substituted nitroaromatic substrates more efficiently, NBDO favors meta-substituted, presumably due to distinct active site residues of the two enzymes. Our data implies, however, that the O2 uncoupling and hydroxylation activity cannot be assessed from simple structure-reactivity relationships. By quantifying O2 uncoupling by Rieske dioxygenases, our work provides a mechanistic link between contaminant biodegradation, the generation of reactive oxygen species, and possible adaptation strategies of microorganisms to the exposure of new contaminants.
Bopp, C. E.; Bernet, N. M.; Kohler, H.-P. E.; Hofstetter, T. B. (2022) Elucidating the role of O2 uncoupling in the oxidative biodegradation of organic contaminants by Rieske non-heme iron dioxygenases, ACS Environmental Au, 2(5), 428-440, doi:10.1021/acsenvironau.2c00023, Institutional Repository

Substrate-specific coupling of O2 activation to hydroxylations of aromatic compounds by rieske non-heme iron dioxygenases

Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme FeII center, elementary steps leading to O2 activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O2 activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O2 uncoupling for a series of substituted nitroaromatic compounds with 18O and 13C kinetic isotope effects of dissolved O2 and aromatic substrates, respectively, we show that O2 uncoupling occurs after the rate-limiting formation of FeIII-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O2 uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O2 uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O2 activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases.
Pati, S. G.; Bopp, C. E.; Kohler, H.-P. E.; Hofstetter, T. B. (2022) Substrate-specific coupling of O2 activation to hydroxylations of aromatic compounds by rieske non-heme iron dioxygenases, ACS Catalysis, 12(11), 6444-6456, doi:10.1021/acscatal.2c00383, Institutional Repository

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