Version française
Institutes/ Institute of Biology and Technology Saclay (iBiTec-S)/ Units/ Bioenergetics, structural biology, and mechanisms (SB2SM/URA2096 CNRS)/ High field electronic paramagnetic resonance (LMESS)/ Metalloproteins and protein radicals (A. Ivancich)

Metalloproteins and protein radicals

Anabella IVANCICH
CEA Saclay/Bât. 532

Tél : 01 69 08 28 42
anabella.ivancich@cea.fr


iBiTec-S / SB2SM / LMESS


Moyens humains
Anabella IVANCICH , Group leader
Bennett STREIT, Postdoctoral Fellow


Thèmes de recherche

Our Research is focussed on understanding the diverse reactivity of heme enzymes, with special emphasis on the protein-based radical species playing the role of alternative intermediates to the heme active site in the catalytic cycle. Our finding of an unprecedented tyrosyl radical intermediate formed in bovine liver catalase led us to a comprehensive characterization of heme mono and bifunctional peroxidases (KatGs) with the primary hypothesis that these enzymes may belong to the restricted group of metalloenzymes using protein-based radicals as redox cofactors in their catalytic cycle, such as ribonucleotide reductase and Photosystem II. The well-known role of catalases and peroxidases in the regulation of oxidative stress through the disproportionation of hydrogen peroxide is related to the porphyrin-based radical intermediate. The existence of alternative reactive intermediates located away from the heme active site can be correlated to selectivity towards substrates that cannot access to the metal site. The interest of unravelling the role of protein-based radical intermediates in these enzymes is based on three main aspects: 1) understanding the catalytic cycle and the substrate specificity, 2) understanding the mechanisms by which enzymes stabilize radicals on defined Tyr or Trp residues in order to use them as alternative oxidation sites; 3) understanding the physicochemical properties of protein-based radical intermediates through model complexes (oxidation potentials, effect of the protein microenvironment on the radical stability). A multidisciplinary approach combining multifrequency (9-285 GHz) EPR spectroscopy, biophysical techniques as well as site-directed mutagenesis and X-Ray crystallography is required, the two latter done in collaboration with the laboratory of Peter Loewen in Canada,Christian Obinger  in Vienna, Andrew Smith  in UK.


Mots-clés
Heme peroxidases, catalases, oxidative stress, tuberculosis, KatG, EPR, spectroscopy.


Publications

A. Ivancich and P. C. Loewen (2012). Electron Transfer in Catalases and Catalase-Peroxidases. In Encyclopedia of Biophysics, Gordon C. K. Ed., Springer-Verlag Berlin Heidelberg, Volume 2, pp. 611-614.

Colin J, Jakopitsch C, Obinger C, Ivancich A. (2010). The reaction of Synechocystis PCC6803 catalase-peroxidase (KatG) with isoniazid investigated by multifrequency (9-285 GHz) EPR spectroscopy. Appl. Magn. Reson. 37, 267-277.

Singh, R., Berry, R. E., Yang, F., Zhang, H., Walker, F. A., Ivancich, A. (2010). Unprecedented peroxidase-like activity of Rhodnius prolixus Nitrophorin 2: Identification of the [FeIV=O Por•]+ and [FeIV=O Por](Tyr38•) intermediates and their role(s) in substrate oxidation. Biochemistry 49, 8857–8872.

Smulevich, G., Feis, A., Howes, B. D., Ivancich, A. (2010). Structure-function relationships in heme peroxidases: New insights from electronic absorption, resonance Raman and multifrequency Electron Paramagnetic Resonance spectroscopies. Handbook of Porphyrin Science (Kadish, K. M., Smith, K. M., Guilard, R. Eds), vol. 6, chapter 31, pp 367-453, World Scientific, Hackensack, NJ.

Belin P, Le Du MH, Fielding A, Lequin O, Jacquet M, Charbonnier JB, Lecoq A, Thai R, Courçon M, Masson C, Dugave C, Genet R, Pernodet JL, Gondry M. (2009). Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 106, 7426-7431.

Colin J, Wiseman B, Switala J, Loewen PC, Ivancich A. (2009). Distinct Role of Specific Tryptophans in Facilitating Electron Transfer or as [Fe(IV)=O Trp(center dot)] Intermediates in the Peroxidase Reaction of Bulkholderia pseudomallei Catalase-Peroxidase: A Multifrequency EPR Spectroscopy Investigation. J Am Chem Soc. 131, 8557-8563.

Smith AT, Doyle WA, Dorlet P, Ivancich A. (2009). Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase.Proc. Natl. Acad. Sci. USA 106, 16084-16089.

Wiseman B, Colin J, Smith AT, Ivancich A, Loewen PC. (2009). Mechanistic insight into the initiation step of the reaction of Burkholderia pseudomallei catalase-peroxidase with peroxyacetic acid. J Biol Inorg Chem. 14, 801-11.

Fielding AJ, Singh R, Boscolo B, Loewen PC, Ghibaudi EM, Ivancich A. (2008). Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy. Biochemistry. 47, 9781-92.

Deemagarn T, Wiseman B, Carpena X, Ivancich A, Fita I, Loewen PC. (2007). Two alternative substrate paths for compound I formation and reduction in catalase-peroxidase KatG from Burkholderia pseudomallei. Proteins.66, 219-228.

Singh R, Switala J, Loewen PC, Ivancich A. (2007).Two [Fe(IV)=O Trp(*)] Intermediates in M. tuberculosis Catalase-Peroxidase Discriminated by Multifrequency (9-285 GHz) EPR Spectroscopy: Reactivity toward Isoniazid. J Am Chem Soc. 129, 15954-63.

Carpena X, Wiseman B, Deemagarn T, Herguedas B, Ivancich A, Singh R, Loewen PC, Fita I. (2006).Roles for Arg426 and Trp111 in the modulation of NADH oxidase activity of the catalase-peroxidase KatG from Burkholderia pseudomallei inferred from pH-induced structural changes Biochemistry.45, 5171-5179.

Hanano A, Burcklen M, Flenet M, Ivancich A, Louwagie M, Garin J, Blee E. (2006).Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif. J Biol Chem.281, 33140-33151.

Jakopitsch C, Obinger C, Un S, Ivancich A. (2006).Identification of Trp106 as the tryptophanyl radical intermediate in Synechocystis PCC6803 catalase-peroxidase by multifrequency Electron Paramagnetic Resonance spectroscopy. J Inorg Biochem.100, 1091-1099.

Carpena X, Wiseman B, Deemagarn T, Singh R, Switala J, Ivancich A, Fita I, Loewen PC. (2005).A molecular switch and electronic circuit modulate catalase activity in catalase-peroxidases. EMBO Rep.6, 1156-1162.

Jakopitsch C, Ivancich A, Schmuckenschlager F, Wanasinghe A, Poltl G, Furtmuller PG, Ruker F, Obinger C. (2004).Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants. J Biol Chem.279, 46082-46095.

Loewen PC, Carpena X, Rovira C, Ivancich A, Perez-Luque R, Haas R, Odenbreit S, Nicholls P, Fita I. (2004).Structure of Helicobacter pylori catalase, with and without formic acid bound, at 1.6 A resolution. Biochemistry.43, 3089-3103.

Ivancich A, Jakopitsch C, Auer M, Un S, Obinger C. (2003).Protein-based radicals in the catalase-peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site. J Am Chem Soc. 125, 14093-14102.

Jakopitsch C, Auer M, Ivancich A, Ruker F, Furtmuller PG, Obinger C. (2003). Total Conversion of Bifunctional Catalase-Peroxidase (KatG) to Monofunctional Peroxidase by Exchange of a Conserved Distal Side Tyrosine. J Biol Chem. 278, 20185-20191.