Oxygen-17 Nuclear Magnetic Resonance Spectroscopic Studies of Carbonmonoxy Hemoproteins

We have obtained surprisingly narrow ¹⁷ O nuclear magnetic resonance (NMR) spectra at 8.45 and 11.7 T (corresponding to ¹⁷ O frequencies of 48.8 and 67.8 MHz) from C ¹⁷ O 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 ¹⁷ O 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 (T ₁ ) measurements that the ¹⁷ O 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 ¹⁷ O QCC values of 0.9 MHz and τ _c of 23 ns (at ω ₀ τ _c = 10). In all cases, our results are consistent with an ¹⁷ O 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 ¹⁷ 0 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 ¹³ C NMR relaxation data, indicate little “internal motion” of the heme-CO group in this system. Our results also demonstrate a linear relationship between the ¹⁷ O NMR chemical shift and v _co , the infrared stretching frequency of the CO ligand, and between the ¹⁷ O chemical shift and the CO binding affinity of the protein. In addition, the ¹⁷ O NMR results are also in good agreement with previous time-differential perturbed γ-ray angular correlation (PAC) results on [ ¹¹¹ In]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.