A copper- containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015 has its own unique blue or type 1 copper protein resonance Raman spectrum in the usual Cu-S-Cys stretching region, nu(Cu-SCys), with a pair of strong peaks at 412 and 420 cm(-1) and a weak peak at 364 cm(-1). The predominantly nu(Cu-SCys) Raman bands at 412, 420, and 364 cm(-1) of the type 1 copper site all shifted to higher frequencies upon binding of nitrite to the type 2 copper site, and the resonance Raman difference spectra progressively intensified with the increments of nitrite ion concentration. Positive support for substrate binding to the type 2 copper is provided by the nu(Cu-SCys) bands in the resonance Raman spectrum of a type 2 copper- depleted enzyme, which is insensitive to the presence of NO2-. The shift to higher frequency of the Raman bands of the type 1 copper center with the addition of nitrite ions suggests a stronger Cu-S-Cys interaction in the substrate-bound A. xylosoxidans nitrite reductase.