The Importance of the Long Type 1 Copper‐Binding Loop of Nitrite Reductase for Structure and Function

Abstract

The long 15-residue type 1 copper-binding loop of nitrite reductase has been replaced with that from the cupredoxin amicyanin (7 residues). This sizable loop contraction does not have a significant effect on the spectroscopy, and therefore, the structures of both the type 1 and type 2 Cu(II) sites. The crystal structure of this variant with Zn(II) at both the type 1 and type 2 sites has been determined. The coordination geometry of the type 2 site is almost identical to that found in the wild-type protein. However, the structure of the type 1 centre changes significantly upon metal substitution, which is an unusual feature for this class of site. The positions of most of the coordinating residues are altered of which the largest difference was observed for the coordinating His residue in the centre of the mutated loop. This ligand moves away from the active site, which results in a more open metal centre with a coordinating water molecule. Flexibility has been introduced into this region of the protein. The 200 mV increase in the reduction potential of the type 1 copper site indicates that structural changes upon reduction must stabilise the cuprous form. The resulting unfavourable driving force for electron transfer between the two copper sites, and an increased reorganisation energy for the type 1 centre, contribute to the loop variant having very little nitrite reductase activity. The extended type 1 copper-binding loop of this enzyme makes a number of interactions that are important for maintaining quaternary structure.

Proton-Coupled Electron Transfer in the Catalytic Cycle of Alcaligenes xylosoxidans Copper-Dependent Nitrite Reductase

“…The slightly altered position of this residue in the substrate-free N90S and N90S–H254F structures (vide infra) suggests that this change could be the “trigger” for the loop movement. Notably this loop has been shown crystallographically to be involved in the formation of a potential electron transfer complex between Cyt 551 and NiR, and mutation and truncation of the residues in this loop causes a loss of catalytic activity and 200 mV increase in redox potential of the T1Cu site in Ax NIR . This altered loop conformation in the substrate-bound N90S structure introduces a hydrogen bond network between Ser90 and the nearby Glu133 and Asn107 residues.…”

Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis

“…The importance of the cupredoxin scaffold in defining the orientation of the Cys and Met ligands located at the start and end respectively of the engineered loop has been already documented in elegant loop mutant studies of azurin, 55 , 58 plastocyanin, pseudoazurin 51 and nitrite reductase. 57 These studies describe the role of T1-containing scaffolds upon the metal site. Here we show that the Cu A -containing scaffold plays a similar role in defining the position of the Cys and Met ligands, even when engineering a shortened loop that lacks the extra Cys required for the Cu A site.…”

Bioinorganic chemistry

“…The 15-residues loop in the original enzyme has been replaced with a 7-residues loop (from cupredoxin amicyanine) and the effect of this on the enzyme's structure and activity studied by spectroscopic and structural means. 42 A series of model compounds have been reported with the aim of gaining insight into the reaction mechanism of copper nitrite reductases. The copper(I) complexes are based on tridentate tris(4-imidazolyl)carbinol ligands with bulky substituents.…”