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Accueil > Les équipes > Biophysique des métalloprotéines - BIP07 > Thèmes de Recherche > Reactivity of complex molybdoenzymes

Reactivity of complex molybdoenzymes

par grimaldi - publié le , mis à jour le

Prokaryotic molybdoenzymes are involved at key steps of global biogeochemical cycles and contribute at metabolizing various toxic compounds. They participate in biological processes having a strong socioeconomic or environmental interest : denitrification, CO2 production or trapping in relation to the greenhouse effect, cleanup of wasted water and soils. In the current research context related to issues of sustainable development, capabilities of molybdenum enzymes are an important element for setting up biotechnological processes of great interest.

In this context, we study bacterial molybdoenzymes, such as those catalyzing the dissimilatory reduction of nitrate into nitrite, i.e. the dimeric periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides and the trimeric membrane-bound nitrate reductase from Escherichia coli. We use a multidisciplinary approach to provide an integrated understanding of the catalytic mechanisms of these two enzymes, including all constitutive processes : catalysis at the active site, proton and electron transfer, intramolecular transport of substrate. We pay a particular attention to identify the molecular factors driving substrate selectivity, reactivity and directionality of the catalysed reaction. This approach is also of great value for understanding evolutionary processes leading to molecular adaptation of these enzymes to changing environments or substrates.

Finally, we work at elucidating at the molecular level the mechanisms that ultimately lead to the formation of active molybdoenzymes during their biosynthesis, with particular attention to the role of dedicated chaperones involved in molybdoenzyme maturation.

Active site of the membrane-bound nitrate reductase A (NarGHI) from E. coli. The molybdenum atom is shown in green. A Fe4S4 cluster having peculiar EPR spectroscopic properties (S = 3/2) is located in the vicinity of the molybdenum cofactor.

For these studies, we combine several spectroscopic and theoretical methods :

  • continuous wave and pulsed multifrequency (S, X and Q-band) EPR spectroscopy
  • Redox potentiometry
  • Optical and magneto-optical spectroscopy (MCD)
  • Theoretical calculations (DFT)
  • Freeze-Quench techniques

Collaborations :

  • Axel Magalon & Anne Walburger, LCB, Marseille
  • David Pignol, Pascal Arnoux & Monique Sabaty, LBC, CEA - Cadarache
  • Christophe Léger, Vincent Fourmond & Sébastien Dementin, BIP, Marseille
  • Wolfgang Nitschke & Barbara Schoepp-Cothenet, BIP, Marseille
  • Marius Réglier, Olga Iranzo, Renaud Hardré & Maylis Orio, ISM2, Marseille

Funding :

  • French National Research Agency (ANR), Project Molyere 2016-2020
  • Aix-Marseille University, Amidex 2015-2017
  • CNRS, Mission pour l’Interdisciplinarité 2014-2015
  • French National Research Agency (ANR), Project MC2 2011-2015
  • French National Research Agency (ANR), Project ERMoE 2006-2010,

Publications (since 2003) :

  • J. Rendon, F. Biaso, P. Ceccaldi, R. Toci, F. Seduk, A. Magalon, B. Guigliarelli & S. Grimaldi
    Elucidating the structures of the low- and high-pH Mo(V) species in respiratory nitrate reductase : A combined EPR, 14,15N HYSCORE, and DFT study
    Inorg. Chem. (2017), 56, 4422-4434
  • S. Grimaldi, F. Biaso, B. Burlat & B. Guigliarelli
    Electron Paramagnetic Resonance studies of molybdenum enzymes
    In Molybdenum and Tungsten Enzymes : Spectroscopic and Theoretical Investigations
    Edited by Russ Hille, Carola Schulzke & Martin Kirk
    Royal Society of Chemistry Metallobiology Series No. 7 (2016), pp. 68-120
  • R. Arias-Cartin, P. Ceccaldi, B. Schoepp-Cothenet, K. Frick, J.-M. Blanc, B. Guigliarelli, A. Walburger, S. Grimaldi, T. Friedrich, V. Receveur-Bréchot & A. Magalon
    Redox cofactor insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism
    Scientific Reports (2016), 6:37743
  • S. Duval, J. M. Santini, D. Lemaire, F. Chaspoul, M. J. Russel, S. Grimaldi, W. Nitschke & B. Schoepp-Cothenet
    The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase
    Biochim. Biophys. Acta - Bioenergetics (2016), 1857, 1353-1362
  • P. Ceccaldi, J. Rendon, C. Léger, R. Toci, B. Guigliarelli, A. Magalon, S. Grimaldi & V. Fourmond
    Reductive activation of E. coli respiratory nitrate reductase
    Biochim. Biophys. Acta - Bioenergetics (2015), 1847, 1055-1063
  • J. G.J. Jacques, B. Burlat, P. Arnoux, M. Sabaty, B. Guigliarelli, C. Léger, D. Pignol, V. Fourmond
    Kinetics of substrate inhibition of periplasmic nitrate reductase
    Biochim. Biophys. Acta - Bioenergetics (2014), 1837, 1801–1809
  • J. G.J. Jacques, V. Fourmond, P. Arnoux, M. Sabaty, E. Etienne, S. Grosse, F. Biaso, P. Bertrand, D. Pignol, C. Léger, B. Guigliarelli, B. Burlat,
    Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion
    Biochim. Biophys. Acta - Bioenergetics (2014), 1837, 277–286
  • M. Sabaty, S. Grosse, G. Adryanczyk, S. Boiry, F. Biaso, P. Arnoux, D. Pignol
    Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis
    BMC Biochemistry (2013), 14, 28
  • S. Grimaldi, B. Schoepp-Cothenet, P. Ceccaldi, B. Guigliarelli, A. Magalon
    The prokaryotic Mo/W-bisPGD enzymes family : A catalytic workhorse in bioenergetics
    Biochim. Biophys. Acta - Bioenergetics (2013), 1827, 1048–1085
  • M. Lorenzi, L. Sylvi, G. Gerbaud, E. Mileo, F. Halgand, A. Walburger, H. Vezin, V. Belle, B. Guigliarelli, A. Magalon
    Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone
    Plos one (2012), 7, e49523
  • F. Biaso, B. Burlat, B. Guigliarelli
    DFT Investigation of the Molybdenum Cofactor in Periplasmic Nitrate Reductases : Structure of the Mo(V) EPR-Active Species
    Inorg. Chem. (2012), 51, 3409–3419
  • V. Fourmond, B. Burlat, S. Dementin, M. Sabaty, P. Arnoux, E. Etienne, B. Guigliarelli, P. Bertrand, D. Pignol, C. Léger
    Dependence of Catalytic Activity on Driving Force in Solution Assays and Protein Film Voltammetry : Insights from the Comparison of Nitrate Reductase Mutants
    Biochemistry (2010), 49, 2424–2432
  • V. Fourmond, B. Burlat, S. Dementin, P. Arnoux, M. Sabaty, S. Boiry, B. Guigliarelli, P. Bertrand, D. Pignol, C. Léger
    Major Mo(V) EPR Signature of Rhodobacter sphaeroides Periplasmic Nitrate Reductase Arising from a Dead-End Species That Activates upon Reduction. Relation to Other Molybdoenzymes from the DMSO Reductase Family
    J. Phys. Chem. B (2008), 112, 15478-15486
  • P. Lanciano, A. Savoyant, S. Grimaldi, A. Magalon, B. Guigliarelli & P. Bertrand*
    A new method for the spin quantitation of [4Fe-4S]1+ clusters with S = 3/2. Application to the FS0 center of the NarGHI nitrate reductase from Escherichia coli
    J. Phys. Chem. B (2007), 111, 13632-13637
  • P. Lanciano, A. Vergnes, S. Grimaldi, B. Guigliarelli & A. Magalon*
    Biogenesis of a respiratory complex is orchestrated by a single accessory protein
    J. Biol. Chem. (2007), 282, 17468-17474
  • P. Bertrand, B. Frangioni, S. Dementin, M. Sabaty, P. Arnoux, B. Guigliarelli, D. Pignol, C. Léger
    Effects of slow substrate binding and release in redox enzymes : Theory and application to periplasmic nitrate reductase
    J. Phys. Chem. B (2007), 111, 10300-10311
  • S. Dementin, P. Arnoux, B. Frangioni, S. Grosse, C. Leger, B. Burlat, B. Guigliarelli, M. Sabaty, D. Pignol
    Access to the active site of periplasmic nitrate reductase : Insights from site-directed mutagenesis and zinc inhibition Studies
    Biochemistry (2007), 46, 9713-9721
  • B. Frangioni, P. Arnoux, M. Sabaty, D. Pignol, P. Bertrand, B. Guigliarelli, C. Léger
    In Rhodobacter sphaeroides respiratory nitrate reductase, the kinetics of substrate binding favors intramolecular electron transfer
    J. Am. Chem. Soc. (2004), 126, 1328-1329
  • P. Arnoux, M. Sabaty, J. Alric, B. Frangioni, B. Guigliarelli, J. M. Adriano, D. Pignol
    Structural and redox plasticity in the heterodimeric periplasmic nitrate reductase
    Nat. Struct. Biol (2003), 10, 928-934