We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C l7 O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7 → Val E7; His E7 → Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 188.8.131.52) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 184.108.40.206), in some cases as a function of pH, and have determined their isotropic 17 O NMR chemical shifts, δ i , and spin-lattice relaxation times, T 1 . We have also obtained similar results on a picket fence prophyrin, [5, 10, 15, 20-tetrakis(α,α,α,α-pivalamidophenyl)porphyrinato]iron(II) (l-Melm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C–O vibrational frequencies, v(C–O), and δ i , between μ(C–O) and the ,7 O nuclear quadrupole coupling constant (e 2 qQ/h, derived from T 1 ), and as expected between e 2 qQ/h and δ i . Taken together with the work of others on the 13 C NMR of 13 CO-labeled proteins, where we find an excellent correlation between δ i ( 13 C) and v(Fe–C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of π-back-bonding from Fe d to CO π* orbitals, as outlined previously [Li, X.-Y., & Spiro, T. G. (1988) J. Am. Chem. Soc. 110, 6024], The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.
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