The molecular mechanism of copper-containing nitrite reductases

Nitrogen is an essential component of all living systems and availability is largely governed by the terrestrial nitrogen cycle. From this system, several forms of nitrogen are available for use in the synthesis of biologically relevant molecules such as proteins and nucleic acids, and for bioenergetic respiratory processes in facultative anaerobes. An imbalance in the nitrogen cycle can lead to many globally harmful events, including toxification of water sources and production of the environmental pollutants nitric and nitrous oxide. One of the crucial enzymes of the nitrogen cycle is nitrite reductase, which catalyses the key environmental step of converting a mineral form of nitrogen (nitrite) into a gaseous form (nitric oxide). The objective of this thesis is to characterize the molecular mechanism of coppercontaining nitrite reductases (CuNiR) through the identification and characterization of catalytically important residues of the CuNiR from Alcaligenes faecalis S-6 (AfNiR). In the native nitrite-soaked crystal structure of AfNiR, nitrite bound to the active site copper forms a hydrogen bond with the side-chain of Asp98 that is connected to His255 through a solvent-bridged hydrogen bond. Three variants (D98N, H255D and H255N) of Asp98 and His255 were generated to probe the proton donation role of these residues. Nitrite reductase assays showed large reductions in activity for all three variants relative to native AfNiR suggesting an essential catalytic role for Asp98 and His255. Spectroscopic studies showed that the mutations did not affect significantly copper occupation although small changes in the electronic structures of the metal co-factors were detected, consistent with positional shifts in the ligand solvent observed in the structures. High-resolution nitrite-soaked crystal structures of the D98N and H255N AfNiR variants in both the.oxidized and reduced state show clearly that both residues are essential for directing productive nitrite binding in the active site. In the D98N nitrite-soaked structures both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N5 atom of Asn98 to form a hydrogen bond with the bound nitrite indicating that the hydrogen bond between Asp98 and nitrite in the native AfNiR structure is important in anchoring nitrite in the active site for catalysis. In the nitritesoaked H255N crystal structures, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. The reoriented nitrite serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated simultaneously to the copper. In the native enzyme, an isoleucine residue (Ile257) occludes the active site pocket and packs closely against the bound nitrite. A combinatorial mutagenesis approach was used to generate small library of six variants at position 257 in AfNiR. The activities of these six variants span nearly two orders of magnitude with one variant I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. Highresolution (< 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies show that a bidentate, O-coordinate mode of nitrite binding is required for catalytic productivity and that the nature of the residue at position 257 strongly directs this mode of binding.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat