Abstract
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 (approximately 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.
Highlights
It is well recognized that hemoglobins (Hbs)3 are widespread in all phyla
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, the E7 Tyr is seen as displaced toward the globin CD region [18]
Summary
Biological Materials—P. epiclitum and G. crumenifer (Platyhelminthes, Trematoda, Paramphistomidae) parasitic in the rumen of the common. Purification of Trematode Hemoglobins and Sequence Determination—Purification and primary structure determination of the P. epiclitum Hb is described in Rashid et al [17]. Purification and protein sequencing of the G. crumenifer Hb was performed as described for P. epiclitum. The instrumentation and measurement procedures have been described elsewhere [9]. Deoxy (reduced) hemoglobin was prepared by the addition of a small aliquot of dithionite solution under anaerobic conditions. The CO complexes were prepared by the addition of CO (12C16O or 13C18O) to anaerobic solutions of dithionitereduced protein (ϳ70 M) in 100 mM sodium phosphate buffer, pH 7.4 in tightly sealed Raman cells. The 13C18O gas was obtained from ICON (Mt. Marion, NY) and 12C16O was purchased from Matheson (Rutherford, NJ). Optical spectra were recorded before and after Raman measurements to ensure sample integrity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
