Computational, biochemical, and spectroscopic studies of the copper-containing nitrite reductase from the denitrifier Sinorhizobium meliloti 2011

GOMEZ, MA C.; FERRONI, F; DALOSTO, S; BRONDINO, C

Oro Verde

Congreso; 3er. Congreso Argentino de Bioinformática y Biología Computacional; 2012

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.