Multiple Active Site Conformers in the Carbon Monoxide Complexes of Trematode Hemoglobins

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 (ϳ60 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.It is now well recognized that hemoglobins (Hbs) 3 are widespread in all phyla. There has been a renewed interest in understanding the structure and function of Hbs in recent years (1-5), because these Hbs are located in diverse cellular compartments and possess atypical physicochemical properties. Hbs have been discovered in about 33% of the animal species, and in addition to being present in vertebrates, they are found in the phyla that include nematode, mollusc, and annelid, to name but a few. Single subunit globins have been discovered lately in algae, nonsymbiotic plants, and a number of prokaryotes ranging from bacteria to cyanobacteria (see Refs. 2-5). Additionally, flavoproteinbound chimeric Hbs have been found in bacteria (6) and yeast (7). In most cases, the nonvertebrate Hbs are proposed to have dissimilar functions from those in vertebrate Hbs; the latter class is established as consisting of O 2 carrier proteins. However, additional cellular activities have been proposed recently for vertebrate Hbs as well (8).Although the nonvertebrate Hbs bind O 2 and other ligands just as the vertebrate Hbs, the kinetic and structural properties of the O 2 complexes (see for example Refs...

Structural and Functional Properties of a Single Domain Hemoglobin from the Food-borne Pathogen Campylobactor jejuni

“…Consequently, Fe 3ϩ -CN is more flexible than Fe 2ϩ -CO and they respond differently to steric and electrostatic influences exerted by the distal protein matrix surrounding the ligand in hemeproteins. The higher flexibility of the Fe-C-N moiety is consistent with its lower bending frequency (ϳ350 -460 cm Ϫ1 ) with respect to that of the Fe-C-O moiety (ϳ560 -590 cm Ϫ1 ) (34,40). Cyanide-bound ferric complexes of hemeproteins can adopt either "linear" or "bent" conforma- tions.…”

“…The data shown in B are taken from the literature (6,8,10,30,34,(52)(53)(54)(55). L stands for the proximal ligand of the heme iron.…”

The hemoglobins of the trematodes Fasciola hepatica and Paramphistomum epiclitum: A molecular biological, physico‐chemical, kinetic, and vaccination study

“…Beside Fa.he . Hbs, an acetylated N terminus has also been reported for the trematode Gastrotylax cruminifer Hb (Das et al 2006). Acetylated N termini are, in general, scarce in nonvertebrate Hbs, and it is currently not known whether the functional significance of such modification is simply that of protecting the protein in the cytosolic compartment against proteolytic degradation.…”

“…The Hb of Paramphistomum epiclitum ( Pa.ep . ), so far the best characterized of trematode Hbs, is monomeric and displays the major determinants of the typical globin fold (Rashid et al 1997; Kiger et al 1998; Pesce et al 2001; Das et al 2006). The heme‐ligand binding site displays a TyrB10/TyrE7 distal residue pair, and a high oxygen affinity ( P 50 < 0.001 mm Hg).…”

“…The network involves TyrB10, a distal‐site‐trapped water molecule, ThrE10, and the carbonyl group of TyrE7. The TyrE7 side chain, however, is locked next to the CD globin region in a conformation unsuitable for stabilization of the heme‐bound ligand (Zhang et al 1997; Kiger et al 1998; Pesce et al 2001; Das et al 2006).…”