Ligand binding in the ferric and ferrous states of Paramecium hemoglobin

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglob in (Hb) that has only 116 amino acid residues. This Hb shares the simultane ous presence of a distal E7 glutamine and a B10 tyrosine with several inver tebrate Hbs. In the study presented here, we have used ligand binding kinet ics and resonance Raman Spectroscopy to characterize the effect of the dist al pocket residues of Paramecium Hb in stabilizing the heme-bound ligands, In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition ta kes place with a pK(a) of similar to9.0, The oxygen affinity (P-50 = 0.45 T orr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hy drogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy comple x is an equilibrium mixture of a hydrogen-bonded closed structure and an op en structure. Oxygen will dissociate preferentially From the open structure , and therefore, the fraction of open structure population controls the rat e of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the h igher on- and off-rates for CO relative to those in myoglobin, A high rate of ligand binding is also consistent with the observation of an Fe-histidin e stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain, We postulate that the function of Paramecium Hb is to sup ply oxygen for cellular oxidative processes.