UBC Theses and Dissertations
Copper-nitrosyl formation by nitrite reductase Tocheva, Elitza I.
Copper-containing nitrite reductase (NiR) is part of the denitrification pathway employed by bacteria to generate energy from reducing equivalents during low oxygen availability. NiR is located in the periplasm and catalyzes the committed step of denitrification, the one electron reduction of nitrite to nitric oxide. The enzyme is a homotrimer with two spectroscopically distinct copper sites per monomer, classified as type 1 and type 2. The type 1 copper site is located near the surface of the protein and receives electrons from a physiological electron donor, such as pseudoazurin. The electrons are then passed onto the type 2 copper site, which is the catalytic center of NiR. The type 2 copper is located at the bottom of a 16 Å deep cavity at the interface between two adjacent subunits. Efficient electron transfer is ensured by the presence of a Cys-His bridge that connects the two copper sites. A copper-nitrosyl intermediate is formed by NiR during catalysis; however, it has not been characterized previously. To address how NiR may interact with nitric oxide and to better understand the transition between substrate and product binding to the type 2 copper during catalysis, crystal structures and EPR spectra were analyzed of the substrate- and product-bound NiR. In the crystal structure of the substrate-bound NiR from Alcaligenes faecalis S-6 (AfNiR) extended to 1.4 Å resolution, the NO₂⁻ coordinates to the copper primarily by a single oxygen atom (Cu-Oc distance of ~2 Å). In addition, the second oxygen and nitrogen atoms are situated ~2.3 Å from the metal, positioning nitrite almost face-on with respect to the copper. A copper-nitrosyl intermediate forms during the catalytic cycle of AfNiR. To examine a possible copper-NO interaction in AfNiR, crystals of the reduced enzyme were exposed to NO in an anaerobic environment. The x-ray crystal structure to 1.3 Å resolution reveals an NO molecule interacting with the catalytic center. The NO molecule coordinates in a side-on fashion such that both the N and O atoms are equidistant from the copper (~2 Å). The oxidation states of the type 1 and type 2 copper atoms were monitored by EPR spectroscopy. Exposure of the reduced protein solution to NO results in a spectrum indicative of an oxidized type 2 copper site. Observation of NO bound to the catalytic center and measurement of the oxidation state of the metal gave rise to a formal description of the copper-nitrosyl in AfNiR as a Cu(II)-NO⁻. Comparison of the NO₂⁻ -bound with the NO-bound crystal structure of AfNIR provides a plausible explanation of how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The copper-nitrosyl catalytic intermediate formed by AfNiR is formally described as Cu(I)-NO⁺, which is a system that has one less electron than the copper-nitrosyl proposed to be formed by the addition of exogenous NO to reduced AfNiR. Copper-nitrosyl complexes of oxidized wild-type and variant forms of AfNiR were formed by prolonged exposure of crystals to exogenous NO and the structures were determined to 1.8 Å or better resolution. Exposing oxidized wild-type crystals to NO results in the formation of nitrite bound to the type 2 copper. The type 1 copper site variant, H145A, prevents electron exchange with the type 2 site and the reverse reaction is disrupted. Instead, both oxidized and reduced H145A have NO bound side-on to the type 2 copper as observed previously with the reduced wild-type enzyme. Asp98 forms hydrogen bonds to both bound substrate and product. In the D98N variant of AfNiR, NO binding is partially disordered. EPR spectra of each AfNiR-NO complex under similar buffer conditions are indicative of stable copper-nitrosyls that are formally assigned as Cu(II)-NO⁻. Reaction schemes can account for the formation of a stable copper-nitrosyl when starting from either the oxidized or reduced H145A variant. The ability of several small molecules to bind to the type 2 copper of AfNiR and inhibit the enzyme was examined by crystallography and enzyme kinetics. Inhibitors such as nitrate, formate, acetate, nitrous oxide and azide are able to interact with the catalytic center. However, kinetic data showed that tested inhibitors weakly inhibit the enzyme (K[sub i]>2 mM). All inhibitors, except azide, bind ~0.15 Å further away from the copper than either the product or substrate. Azide binds with a Cu-N distance of ~2 Å and shows the strongest inhibition with a K[sub ic] of 2 mM. Mixed inhibition was observed for formate and acetate; however, kinetic data show that nitrate is an uncompetitive inhibitor with respect to nitrite. Through steric clashes and a requirement for a hydrogen bond with Asp98, the active site of NiR is able to favour nitrite binding and discriminate against other small molecules present in the periplasm. Altogether the studies presented in this thesis indicate how AfNIR is specific for its substrates and how this specificity is fine tuned by the active site residues.
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