Copper nitrite reductase contains a T1 and T2 Cu center and catalyzes the one electron reduction of nitrite to NO. This study determines that reaction of the reduced enzyme with excess NO generates the T1 reduced T2 oxidized-nitrite bound form and not the eta(2)-NO center dot Cu+ species observed in the crystal structure. Reaction intermediates have been trapped and N2O gas detected during this reaction, allowing elucidation of the mechanism by which both the T2 and NO are oxidized by the reaction of NO with the fully reduced enzyme. Density functional theory calculations provide insight into factors which favor the eta(2)-NO center dot Cu+ complex in the crystal structure.