We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-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. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)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 delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) 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 pi-back-bonding from Fe d to CO pi* 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.

4. The holoazurin Trp 'Lb absorption band red shifts ca. 1 nm compared to apoazurin, while the 'Bb transition red shifts ca. 2 nm. The hydrophobicity of the Trp environment appears little changed between Cu(I) azurin, Cu(II) azurin, and apoazurin as evidenced by the narrowness of the Trp 'Lb 0-0 bands and from the Raman band shape for the Trp 1354-cm'1 band. The Trp 0-0 'Li, absorption spectral shifts most likely derive from subtle environmental changes, possibly alterations at sites somewhat distant from the Trp ring, such as alterations in electrostatic interaction from the Cu site.5. The absorption difference spectra (oxidized minus reduced as well as holoazurin minus apoazurin) show large changes in the 200-300-nm spectral region which presumably derive from alterations in sulfur and imidazole -* Cu(II) charge-transfer transitions. Numerous charge-transfer transitions can occur since the Cu is ligated to two histidines, one cysteine, and one methionine; simple Cu(II)-(imidazole)4 complexes show increased absorbances throughout the 200-300-nm spectral region (Figure 6).We thus conclude that facile energy transfer between the Trp and the Cu-ligand complex is responsible for the fluorescence quenching in Trp. Presumably this quenching occurs between the Trp 'Laib states and a Cu-ligand charge-transfer state. In addition, the Trp absorption spectral shifts between the Cu(I) azurin, Cu(II) azurin, and apoazurin suggest intimate coupling between the electronic transitions of the Cu-ligand complex and those of Trp. It is possible that strong excitonic interactions are present.

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