Insights into Redox Partner Interactions and Substrate Binding in Nitrite Reductase from Alcaligenes xylosoxidans: Crystal Structures of the Trp138His and His313Gln Mutants^,

Abstract

Dissimilatory nitrite reductase catalyses the reduction of nitrite to nitric oxide within the key biological process of denitrification. We present biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry. Trp138, adjacent to one of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II. Our data reveal that the Trp138His mutant is fully active using methyl viologen as an artificial electron donor, but there is a large decrease in activity using azurin I. These observations together with its crystal structure at a high resolution of 1.6 A confirm the importance of Trp138 in electron transfer and thus in productive interaction with azurin. A "hydrophobic pocket" on the protein surface has been identified as the channel through which nitrite may be guided to the catalytic type-2 Cu site. Glu133 and His313 at the opening of the pocket are conserved among most blue and green copper nitrite reductases (CuNiRs). The failure to soak the substrate into our high-resolution crystal form of native and mutant CuNiRs has been linked to the observation of an extraneous poly(ethylene glycol) (PEG) molecule interacting with His313. We present the crystal structure of His313Gln and the substrate-bound mutant at high resolutions of 1.65 and 1.72 A, respectively. The observation of the substrate-bound structure for the His313Gln mutant and inhibitory studies with PEG establishes the role of the hydrophobic pocket as the port of substrate entry.

Conformation of Pseudoazurin in the 152 kDa Electron Transfer Complex with Nitrite Reductase Determined by Paramagnetic NMR

“…[22][23][24] Similar to previous studies on NiR-Paz interaction, 7,8 the chemical shift map obtained for the complex indicates a binding site close to the exposed copper ligand, His81, which is expected to be involved in the interprotein ET ( Figure 2). 25 …”

“…25,27 A three-to fourfold decrease in the rate of the interprotein ET was observed in Trp and Tyr mutants. In our study, the distance between the equivalent Tyr203 of NiR and His81, the exposed copper ligand in Paz, is about 7 Å and therefore less likely to be part of the path calculated for ET.…”

pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

“…For example, the activity of the enzyme was assessed before crystallization setups by measuring the reoxidation of dithionite-reduced enzyme with nitrite (data not shown). As a further precaution, nitrite was added in 100-fold excess to the protein solution before mixing with precipitant solution to avoid problems with precipitants preventing nitrite binding, which has previously been reported (35). Finally, in the structure at pH 6.0, full occupancy was recovered for the oxygen atoms, and the hypothesis that they may represent two solvent molecules could, hence, be rejected because steric effects would prevent both solvent molecules to be present at the same time.…”

Crystal structures of the nitrite and nitric oxide complexes of horse heart myoglobin

“…A second class of dissimilatory NiR enzymes contain copper in the active site [7]. Recent high-resolution crystal structures of the nitrite adducts of the copper-containing NiR [8] from Alcaligenes xylosoxidans (the His313Gly mutant) [9] and the soil bacterium Achromobacter cycloclastes [10] reveal an asymmetric O,O 0 -bidentate binding of the nitrite ligand (structure II).…”