To probe the active site of copper-containing nitrite reductase (NiR), the carbonyl derivatives of the wildtype enzyme and the Asp98Asn variant were studied by Fourier transform infrared (FTIR) spectroscopy. CO bound to wild-type NiR exhibits a single absorption band at 2050 cm(-1) (Delta nu = similar to 11 cm(-1)) arising from the C-O stretching mode. This band is not significantly affected by pH change (pH less than or equal to 8.0) or by H/D exchange. Interestingly, CO bound to the Asp98Asn variant exhibits two C-O stretching frequencies, one located at 2060 cm(-1) (A(1)) and the other at 2041 cm(-1) (A(2)). These two bands are both temperature and pH dependent, and the equilibrium constant K-298 (1.01), DeltaH degrees (-14(1) kJ/mol), and DeltaS degrees (-45(4) J/mol) for the A(1) -A(2) equilibrium (pH 7.0) were determined. In addition, H/D exchange results in a similar to2 cm(-1) shift of CO to the red for Az but not Al. On the basis of these results and the X-ray crystal structure of NiR, we conclude that the single band at 2050 cm(-1) observed in wild-type NiR is a single, non-hydrogen-bonded C-O stretching mode. The doublet band observed in the Asp98Asn variant indicates that the bound CO occupies two orientations and suggests that the orientation corresponding to band A? participates in hydrogen bonding. These results indicate a role for Asp98 in controlling the orientation and dynamics of ligand binding to the copper center. Implications concerning previous mechanistic arguments arising from analysis of the three-dimensional structure of the enzyme are discussed.