INVESTIGADORES
BRONDINO Carlos Dante
congresos y reuniones científicas
Título:
Computational, biochemical, and spectroscopic studies of the copper-containing nitrite reductase from the denitrifier Sinorhizobium meliloti 2011
Autor/es:
M.C. GOMEZ; F. FERRONI; A.C. RIZZI; S. DALOSTO; C. D. BRONDINO
Lugar:
Oro Verde, Entre Rios
Reunión:
Congreso; 3er. Congreso Argentino de Bioinformática y Biología Computacional; 2012
Resumen:
Nitrite
reductases are enzymes that catalyze the reduction of nitrite to NO in the
denitrification pathway of the biogeochemical nitrogen cycle [1]. In
denitrifying bacteria, this reaction can be catalyzed by two nitrite
reductases, one containing a cd1 heme and the other containing
copper. Copper-containing nitrite reductases (hereafter Nir) present
homotrimeric structure (~ 40 kDa/monomer) with two copper atoms per monomer,
one of type 1 (T1Cu, also blue copper) and other of type 2 (T2Cu, also normal
copper) (Fig. 1). Nirs have been classified into two groups according to the
UV-vis properties of their T1 centers. Blue Nirs exhibit a very intense
absorption band at ~ 590 nm, whereas green Nirs present two intense absorption
bands at ~ 460 and 600 nm. The coordination around both copper centers is shown
in Fig.1b. T1Cu is an electron transfer center, whereas T2Cu is the catalytic
center. The proposed reaction mechanism, which involves a pseudoazurin as external
electron donor (Paz), is schematized in Fig.1.a.
Fig 1- a) Schematic 3D structure of Nir b) Coordination
around T1Cu and T2Cu
We recently
overexpressed and purified the copper containing nitrite reductase from the
denitrifier Sinorhizobium meliloti
2011 (SmNir) [2]. Sinorhizobium meliloti 2011 is a
rhizobia organism which lives symbiotically in root nodules of legumes widely
used in agriculture because of their ability to take dinitrogen from the
atmosphere. We present and discuss the biochemical and spectroscopic properties
of SmNir together with the
computational structural model predicted from its amino acid sequence. We also
report computational studies that describe the
interaction of both types of copper atoms with their
ligands using a classical force field and classical molecular dynamics. The
structure of Nir from Alcaligenes faecalis (pdb accession number, 1SNRB), which shows a high percentage of
identity to SmNir, was used as model.
The force field was addressed using the combination of quantum mechanics (QM)
and classical mechanics (MM) methods known as QM/MM methods [3]. This approach
allowed us to model adequately the active site at the QM level of theory, and
the rest of the system with MM. A total of seven residues, two copper atoms and
one water molecules were treated with QM and the rest, including some water
molecules from the solvent, with Amber force field. We discuss the theoretical
model in terms of the experimental results.