An Experimental and Quantum Chemical Investigation of CO Binding to Heme Proteins and Model Systems:  A Unified Model Based on¹³C,¹⁷O, and⁵⁷Fe Nuclear Magnetic Resonance and⁵⁷Fe Mössbauer and Infrared Spectroscopies

Abstract

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.

The Orientation of CO in Heme Proteins Determined by Time-Resolved Mid-IR Spectroscopy: Anisotropy Correction for Finite Photolysis of an Optically Thick Sample

“…Earlier polarized IR measurements on various heme proteins at ambient temperature [21][22][23]25,26,28,29 reported that the orientation of CO in heme protein is bent at different angles ranging from 15 o to 35 o , similar to earlier crystal structure. 5 However, using the same polarized IR spectroscopy, we have recently found that CO in Mb is almost linear, bent less than 7 o relative to the heme plane normal, 15 consistent with more recent higher resolution X-ray crystal structure 16 and spectroscopic data in solution, 30 thereby establishing necessity of newer paradigm for how Mb discriminate against CO. Even though the theory for determining angles with photoselection spectroscopy is rigorous, there are numerous experimental pitfalls.…”

NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin

“…An upright, perpendicular geometry of the CO group is indicated by time-resolved IR polarization spectroscopy (36) of MbCO, as well as a joint analysis of NMR, 57 Fe Mössbauer, and IR data, using density functional theory (37). Any remaining discrepancy between this model and recent x-ray structures is reconciled by density functional theory calculations (38), which show that the transition dipole of the C-O stretching IR band is not coincident with the C-O bond vector, but lies between the Fe-C bond vector and the heme perpendicular.…”

Solid-State17O NMR as a Probe for Structural Studies of Proteins in Biomembranes

“…8,9 There has been much less 17 O NMR reported from organic materials since 17 O presents even more of a challenge due to the larger quadrupole interaction and, hence, larger line widths. 19 Recent reports of 17 O NMR from biologically relevant materials have included inorganic molecules interacting with hemeproteins, 10 polypeptides, 11,12 amino acids, 2 and nucleic acid bases. 13 Here, we report the first example of 17 O NMR spectra from a selectively labeled transmembrane peptide, 17 O-[Ala12]-WALP23, as a lyophilized powder and incorporated in hydrated vesicles, opening up new possibilities for applications of 17 O solid-state NMR on real biological systems.…”