Oxygen-17 Nuclear Magnetic Resonance Spectroscopic Studies of Carbonmonoxy Hemoproteins

We have obtained surprisingly narrow ¹⁷O nuclear magnetic resonance (NMR) spectra at 8.45 and 11.7 T (corresponding to ¹⁷O frequencies of 48.8 and 67.8 MHz) from C¹⁷O ligands bound to aqueous ferrous myoglobin from Physeter catodon (sperm whale MbCO), from adult human ferrous hemoglobin (HbCO A), and from ferrous hemoglobin from Oryctolagus cuniculus (rabbit HbCO). The ¹⁷O NMR signals from these hemoproteins are not only narrower than anticipated, but in the case of sperm whale MbCO the line shape is distinctly non-Lorentzian. We have thus used the dispersion versus absorption (DISPA) plot method to investigate the origin of these unusual line widths and line shapes and demonstrate that they originate from the multiexponential nature of quadrupolar relaxation outside of the “extreme-narrowing” limit (ω0τC> 1). We find from the DISPA analysis and from spin-lattice relaxation time (T₁) measurements that the ¹⁷O nuclear quadrupole coupling constant (QCC) for sperm whale MbCO is 0.95 MHz, and the rotational correlation time, τ_c, is 14 ns (at ω0τC= 5.8). This indicates a rigid heme-CO unit in sperm whale MbCO, Applying the same type of analysis to human HbCO yields ¹⁷O QCC values of 0.9 MHz and τ_c of 23 ns (at ω₀τ_c = 10). In all cases, our results are consistent with an ¹⁷O chemical shift anisotropy (σ_∥- σ_⊥) value of about 800 ppm for the CO ligand. These results are important for several reasons: first, they represent the first observation of high-resolution ¹⁷0 NMR spectra of the CO ligands in metalloproteins. Second, they represent the first experimental demonstration of multiexponential relaxation of a spin I = ⁵/₂ nucleus and its complete analysis with relaxation theory. Third, our results on sperm whale MbCO, taken together with ¹³C NMR relaxation data, indicate little “internal motion” of the heme-CO group in this system. Our results also demonstrate a linear relationship between the ¹⁷O NMR chemical shift and v_co, the infrared stretching frequency of the CO ligand, and between the ¹⁷O chemical shift and the CO binding affinity of the protein. In addition, the ¹⁷O NMR results are also in good agreement with previous time-differential perturbed γ-ray angular correlation (PAC) results on [¹¹¹In]myglobin and -hemoglobin (Marshall, A. G.; Lee, K. M.; Martin, P. W. J. Am. Chem. Soc. 1980, 102, 1460), and some molecular interpretations of the NMR and PAC results are offered.