1257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes. The mutant has no enzyme activity. Its crystal structure determined at 1.65 Angstrom resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the Nepsilon atom on the imidazole ring of its coordinated residue His 135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant 1257E is in a state corresponding to a transition state in the enzymatic reaction. (C) 2003 Elsevier Science (USA). All rights reserved.