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.
ASJC Scopus subject areas
- Colloid and Surface Chemistry