Nitrite Reductase Activities of Azurin Variants

Abstract

Nitrite reductase (NiR) catalyzes the reduction of nitrite to nitric oxide in the denitrification stage of nitrogen cycle. To study the structure and function of this dinuclear copper enzyme, mimics were designed by adding a second copper-binding site on the surface of mononuclear copper protein azurin. The existing type1 (T1) copper-binding site in azurin is structurally and functionally similar to that of NiR. T1 site was maintained in azurin whereas an additional catalytic type2 (T2) site, similar to the T2 site of NiR, was designed. The designed T2 site in azurin mimics the spectroscopic and functional aspects of NiR. The UV-Vis absorption and EPR spectra of azurin variants showed similarities to that of native NiR. We further examined the activity of our variants using the activity assays. Reduction and re-oxidation assays of T1 site in azurin variants were determined and compared with variants with modified T1 copper center redox potentials.