Resonance Raman studies of the heme–ligand active site of hemoglobin I from Lucina pectinata

Hemoglobin I from the clam Lucina pectinata has the unusual ability to bind hydrogen sulfide. This sulfide-reactive hemoglobin has a high content of phenyl residues in the heme pocket that may account for its ligand binding properties. To confirm this, resonance Raman spectroscopy was used to determine the heme structure of deoxy, oxy, carbon monoxy, metaquo, metcyano, and methydrogen sulfide hemoglobin I (HbI) complexes. The oxidation (ν₄), spin (ν₃), and coordination (ν₂) markers were identified in the high-frequency spectra of all the HbI complexes. The data indicated that the aromatic environment near the heme does not affect the iron oxidation state, coordination state, spin state, and core size marker vibrational modes. The marker bands also revealed that metsulfide HbI complex has an Fe(III), six-coordinate, and low-spin structure. The low-frequency vibrational frequencies for the methydrogen sulfide, metcyano, oxy, and carbon monoxy HbI derivatives showed νFe–S at 374 cm⁻¹, νFe–C at 448 cm⁻¹, νFe–O at 563 cm⁻¹, and νFe–C 516 cm⁻¹, respectively. These results suggest a model where the phenyl residues in the Phe29(B10) and Phe68(E11) positions have strong electrostatic interactions with the metcyano, oxy, and carbon monoxy ligands of the HbIO₂, HbICO, and HbICN complexes. The multipolar interaction explains the higher νFe–C frequency for the carbon monoxy HbI complex, and the lower νCO, νFe–O₂ and νFe–C frequencies for the HbICO, HbIO₂, and HbICN complexes, respectively. The repulsion between the carbonyl group of Gln64(E7) and oxygen or nitrogen of the oxy, carbon monoxy, and metcyano HbI complexes would also contribute to the above behavior. This model implies that Gln64(E7), in HbI Lucina pectinata, does not rotate from its original position to stabilize, by means of hydrogen bonding, the coordination of the other ligands, for example, the metcyano, oxy, and carbon monoxy heme complexes. Instead the electronic interaction between the phenylalanine in B10 and E11 positions stabilize these HbI complexes. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 289–301, 1999