ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
O27. The Major EPR Signature of Periplasmic Nitrate Reductases Arises
from a Species that Activates Upon
V. Fourmond a , B. Burlat a , S. Dementin a , P. Arnoux b , M. Sabaty b , S. Boiry b ,
B. Guigliarelli a , P. Bertrand a , D. Pignol b , C. Léger a
a BIP, CNRS, 31, ch Joseph Aiguier, 13009, Marseille, France
e-mail: vincent.fourmond@ibsm.cnrs-mrs.fr
b Laboratoire de Bioénergétique Cellulaire, Instit, CEA,, 13108, St Paul-lèz-Durance, France
Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze
a variety of oxo-transfer reactions[1]. Regarding nitrate reductases, which are among the most studied members
of this family, much functional information has been gained from EPR spectroscopy[2, 3, 4], but this technique
is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to
active species is still unsure. We use site-directed mutagenesis, EPR and protein film voltammetry[5] to
demonstrate that the MoCo in Rh. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible
reductive activation process that correlates with the disappearance of the so-called "high-g" MoV EPR signal.
Therefore, this most intense and commonly observed signature of the MoCo arises from an inactive state that
gives a catalytically competent species only after reduction. This proceeds, even without substrate, according to
a reduction followed by an irreversible non-redox step, both of which are pH independent. An apparently similar
process occurs in other nitrate reductases (both assimilatory and membrane-bound[6]) and this also recalls the
redox cycling procedure which activates DMSO reductases and simplifies their spectroscopy[7].
References:
[1] Hille, R. Trends in Biochemical Sciences 2002, 27, 360-367.
[2] Butler, C. S.; Charnock, J. M.; Garner, C. D.; Thomson, A. J.; Ferguson, S. J.; Berks, B. C.; Richardson, D. J.
Biochem. J. 2000, 352, 859-864.
[3] Arnoux, P.; Sabaty, M.; Alric, J.; Frangioni, B.; Guigliarelli, B.; Adriano, J.-M.; Pignol, D. Nat. Struct. Mol.
Biol. 2003, 10, 928-934.
[4] Gonzàlez, P.; Rivas, M.; Brondino, C.; Bursakov, S.; Moura, I.; Moura, J. J. Biol. Inorg. Chem. 2006, 11,
609-616.
[5] Léger, C.; Bertrand, P. Chem. Rev., in press.
[6] Field, S. J.; Thornton, N. P.; Anderson, L. J.; Gates, A. J.; Reilly, A.; Jepson, B. J. N.; Richardson, D. J.;
George, S. J.; Cheesman, M. R.; Butt, J. N. Dalton Trans. 2005, 3580-3586.
[7] Bray, R.; Adams, B.; Smith, A.; Bennett, B.; Bailey, S. Biochemistry 2000, 39, 11258-11269.
_____________________________________________________________________
114