Density functional theory calculations were used to investigate the binding mode of copper(I) nitrosyl (Cu(I)-NO) in copper nitrite reductase (CuNIR). The end-on Cu(I)-NO geometry (2) was found to be the global energy minimum, while the side-on binding mode (1) corresponds to a local minimum. Isoleucine-257 severely interacts sterically with the Cu(I)-NO unit when bound end-on but not in the side-on case. In addition, the side-on geometry is also stabilized by a hydrogen bond between aspartic acid-98 and NO, estimated to be similar to 3 kcal/mol. The steric constraint of the CuNIR active site is mainly responsible for the observed side-on coordination of NO in the CuNIR crystal structure. We speculate that a small conformational change of the active site that slightly changes the position of isoleucine-257 would allow NO to bind end-on. This explains the observed end-on binding of NO to copper(l) when CuNIR is in solution.