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

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P135. New Crystallographic Data Provide New Insights in the Catalytic
Mechanism of Nitrate Reduction: Mo or Ligand-based Redox Chemistry?
S. Najmudin a , C. Coelho a , J. Trincão a , P.J. González a , C. Brondino c , I. Moura a , J.J.G. Moura a ,
C.C. Romão b and M. J. Romão a
a
REQUIMTE/CQFB, Departamento de Química, FCT-UNL, 2829-516 Monte de Caparica, Portugal.
b
Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, Av da República, EAN, 2780-
157, Oeiras, Portugal
c
Departamento de Física, Universidad Nacional del Litoral, 3000ZAA Santa Fe, Argentina.
e-mail: mromao@dq.fct.unl.pt
Nitrate reductases are key enzymes present in the biological cycle of nitrogen and catalyze the conversion of
nitrate to nitrite. We have performed extensive crystallographic studies on two periplasmic nitrate reductases: the
enzyme from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harbouring a
bis-molybdopterin guanine dinucleotide (bis-MGD) active site and a [4Fe-4S] cluster and its crystal structure was
solved to 1.9Å resolution [1, 2] while Nap from Cupriavidus necator (CnNapAB) comprises a 91 kDa catalytic
subunit (NapA) and a di-haem c-type cytochrome 17 kDa subunit (NapB) involved in electron transfer. The
CnNapAB crystal structure was solved to atomic resolution (1.5 Å) [3]. For both Naps, crystals were prepared in
different conditions when reacted with reducing agents, substrate or inhibitors and the corresponding structures
were solved.
The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on
that basis, the sixth Mo ligand, originally proposed to be an OH ligand, was clearly assigned as a sulfur atom after
refinement and analysis of the B-factors of all the structures. This unexpected result was confirmed by
independent means. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the
Sγ atom of the Mo ligand CysA140 was substantially shorter than the van der Waals contact distance and varies
between 2.2 Å and 2.85 Å, indicating a partial disulfide bond:
Mo S
This new and unexpected coordination sphere of Mo derived from our studies led us to revise the currently
accepted reaction mechanism for nitrate reductases. Proposals for a new mechanism are discussed taking into
account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry
based solely on the Mo atom. We propose that reduction of nitrate to nitrite involves a combined Mo and ligandbased
redox chemistry of the active site probably via a sulfoxide-ligated Mo-S=O species, instead of the currently
accepted redox chemistry based solely on the Mo atom in the redox cycle of the enzyme. These results show that
distinct aspects attributed to the chemistry of Mo inorganic complexes to date, can also occur in mononuclear Moenzymes
of the DMSO reductase family, opening new research directions in the study of these proteins.
Acknowledgement:.
This work was supported by projectsPOCI/QUI/57641/2004 and PTDC/QUI/64733/2006 financed by the program
POCI2010 and co-financed by FEDER.
References:
[1] Dias et al. Structure, 1999, 7, 65-79.
[2] Coelho et al. Acta Cryst. 2007, F63, 516-519.
[3] Najmudin et al, J Biol Inorg Chem. 2008 Jun;13(5):737-53
_____________________________________________________________________
254
S
S
RS SR
Mo VI
S
Cys
S
S
RS SR
Mo V
RS SR
Mo IV