We have investigated the question of how CO ligands bind to iron in metalloporphyrins and metalloproteins by using a combination of nuclear magnetic resonance (NMR), 57Fe Mössbauer, and infrared spectroscopic techniques, combined with density functional theoretical calculations to analyze the spectroscopic results. The results of 13C NMR isotropic chemical shift, 13C NMR chemical shift anisotropy, 17O NMR isotropic chemical shift, 17O nuclear quadrupole coupling constant, 57Fe NMR isotropic chemical shift, 57Fe Mössbauer quadrupolar splitting, and infrared measurements indicate that CO binds to Fe in a close to linear fashion in all conformational substates. The 13C-isotropic shift and shift anisotropy for an Ao substate model compound:  Fe(5,10,15,20-tetraphenylporphyrin)(CO)(N-methylimidazole), as well as the 17O chemical shift, and the 17O nuclear quadrupole coupling constant (NQCC) are virtually the same as those found in the Ao substate of Physeter catodon CO myoglobin and lead to most probable ligand tilt (τ) and bend (β) angles of 0° and 1° when using a Bayesian probability or Z surface method for structure determination. The infrared νCO for the model compound of 1969 cm-1 is also that found for Ao proteins. Results for the A1 substate (including the 57Fe NMR chemical shift and Mössbauer quadrupole splitting) are also consistent with close to linear and untilted Fe−C−O geometries (τ = 4°, β = 7°), with the small changes in ligand spectroscopic parameters being attributed to electrostatic field effects. When taken together, the 13C shift, 13C shift anisotropy, 17O shift, 17O NQCC, 57Fe shift, 57Fe Mössbauer quadrupole splitting, and νCO all strongly indicate very close to linear and untilted Fe−C−O geometries for all carbonmonoxyheme proteins. These results represent the first detailed quantum chemical analysis of metal−ligand geometries in metalloproteins using up to seven different spectroscopic observables from three types of spectroscopy and suggest a generalized approach to structure determination.

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