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The reductive reaction mechanism of tobacco nitrite reductase derived from a combination of crystal structures and ultraviolet–visible microspectroscopy
Author(s) -
Nakano Shogo,
Takahashi Misa,
Sakamoto Atsushi,
Morikawa Hiromichi,
Katayanagi Katsuo
Publication year - 2012
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24094
Subject(s) - chemistry , ligand (biochemistry) , molecule , nitrite , crystal structure , nitrite reductase , bond cleavage , photochemistry , active site , reaction mechanism , cleavage (geology) , crystallography , stereochemistry , catalysis , organic chemistry , nitrate , biochemistry , receptor , geotechnical engineering , fracture (geology) , engineering
Assimilatory nitrite reductase (aNiR) reduces nitrite to an ammonium ion and has siroheme and a [Fe 4 S 4 ] cluster as prosthetic groups. A reaction mechanism for Nii3, an aNiR from tobacco, is proposed based on high resolution X‐ray structures and UV–Vis (ultraviolet–visible) microspectroscopy of Nii3‐ligand complexes. Analysis of UV–Vis spectral changes in Nii3 crystals with increasing X‐ray exposure showed prosthetic group reductions. In Nii3‐NO 2 −structures, X‐ray irradiation enhanced the progress of the reduction reaction, and cleavage of the NO bond was observed when X‐ray doses were increased. Crystal structures of Nii3 with other bound ligands, such as Nii3‐NO and Nii3‐NH 2 OH, were also determined. Further, by combining information from these Nii3 ligand‐bound structures, including that of Nii3‐NO 2 − , with UV–Vis microspectral data obtained using different X‐ray doses, a reaction mechanism for aNiR was suggested. Cleavage of the two NO bonds of nitrite was envisaged as a two‐step process: first, the NO bond close to Lys224 was cleaved, followed by cleavage of the NO bond close to Arg109. X‐ray structures also indicated that aNiR‐catalyzed nitrite reduction proceeded without the need for conformation changes in active site residues. Geometrical changes in the ligand molecules and the placement of neighboring water molecules appeared to be important to the stability of the active site residue interactions (Arg109, Arg179, and Lys224) and the ligand molecule. These interactions may contribute to the efficiency of aNiR reduction reactions. Proteins 2012; © 2012 Wiley Periodicals, Inc.