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Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite Reductase by Small Molecules

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journal contribution
posted on 15.04.2008, 00:00 by Elitza I. Tocheva, Lindsay D. Eltis, Michael E. P. Murphy
The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 (AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme (Ki >50 mM), the Cu−O distances in these AfNiR−inhibitor complexes were ∼0.15 Å longer than that observed in the AfNiR−nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR−acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly (Kic = 2.0 ± 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu−Nc distance of ∼2 Å, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.