Roles of Trp144 and Tyr203 in copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013

The roles of the Trp144 and Tyr203 residues near the type 1 Cu site of Achromobacter cycloclastes nitrite reductase (AcNIR) have been examined with mutants of AcNIR. Tyr203 is located on the protein surface near the type 1 Cu site of AcNIR, and Trp144 is between the Tyr203 and the type 1 Cu center in AcNIR. Single mutation of Trp144 or Tyr203 in AcNIR to Leu resulted in decreased rate constants of intermolecular electron transfer from its cognate pseudoazurin (AcPAZ) (kET = 1.9 × 105, 2.2 × 105, and 7.3 × 105 M−1 s−1 for W144L, Y203L, and wild-type AcNIR, respectively). The intermolecular electron transfer rate constant of double mutant AcNIR (W144L/Y203L) was the same as those of single mutants (kET = 1.9 × 105 M−1 s−1 for W144L/Y203L). The redox potentials, coordination structures of the type 1 Cu, and the enzyme activities of AcNIR were affected little by the mutation.