Spectroscopic and electrochemical properties of the Met86Gln mutant of Achromobacter cycloclastes pseudoazurin

Abstract

The mutant replacing the Met86 ligand of Achromobacter cycloclastes pseudoazurin (Ac-pAz) with Gln has been prepared and spectroscopically and electrochemically characterized. Ac-pAz has four ligands (2His, Cys, and Met) and donates one electron to its cognate Cu-containing nitrite reductase (Ac-NIR). The mutant ([Met86Gln]pAz) shows the electronic absorption and CD spectra considerably similar to those of zucchini mavicyanin (Mv) and lacquer and cucumber stellacyanins (St) having 2His, Cys, and Gln. The EPR signal of the mutant has an axial character, although those of Mv and St show rhombic signals. The findings indicate that the Cu site having Gln might be a distorted trigonal geometry. The half-wave potentials ( E 1/2) of [Met86Gln]pAz and the intermolecular electron-transfer rate constant ( k ET) from the mutant to Ac-NIR were determined by cyclic voltammetry at pH 7.0 and 25°C. The E 1/2 is +134 mV (versus NHE) and the coordination of Gln instead of Met negatively shifts the E 1/2 of Ac-pAz (+260 mV (versus NHE)). The k ET of [Met86Gln]pAz (1.2×10 6 M −1 s −1) is larger than that of the recombinant Ac-pAz (7.5×10 5 M −1 s −1).