| Sumario: | The properties of CO-inhibited Azotobader vinelandii (Av) Mo-nitrogenase (Nase) 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 mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q_ N2ase variant. In the frequency region above 450 cm, additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by CO isotope shifts). The EXAFS for wild-type Nase 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 A 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 a-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 reactivity of Nase are discussed.
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