Multiple active site conformers in the carbon monoxide complexes of trematode hemoglobins

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (∼60 cm^-1) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm^-1, for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm^-1, respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm^-1, respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.