Intersite structural rearrangement of the blue copper site induced by substrate binding: spectroscopic studies of a copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015.

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-S(Cys)), with a pair of strong peaks at 412 and 420 cm(-1) and a weak peak at 364 cm(-1). The predominantly nu(Cu-S(Cys)) 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-S(Cys)) 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.