The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N(2)ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm(1) mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the alpha-H195Q N(2)ase variant. In the frequency region above 450 cm(1), additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by (CO)-C-13 isotope shifts). The EXAFS for wild-type N(2)ase shows evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 angstrom in the resting enzyme. A DFT model with both a terminal -CO and a partially reduced -CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the alpha-H195Q variant NRVS. The calculations also shed light on the vibrational shake modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. Implications for the CO and N-2 reactivity of N(2)ase are discussed.