Opossum hemoglobin assumes a T quaternary structure upon NO ligation in the absence of organic phophates at pH 6.7. In addition, stripped opossum hemoglobin exhibits a low oxygen affinity when compared to human hemoglobin and a pH-dependent heme-heme interaction with an n value of 2.14 at pH 7.0 and 2.46 at pH 7.35. These observations indicate that opossum hemoglobin may have a destabilized oxy structure when compared to hemoglobin A due to differences in primary structure. Thus, the strong trans ligand effect of nitric oxide is able to disrupt the proximal histidine-iron bond in the alpha-hemes triggering a conformational transition to the T state. Absence of a distal histidine in the alpha-subunits and, therefore an impaired donor acceptor interaction with the sixth ligand, could contribute to the lack of stability of the R quaternary structure in opossum nitrosylhemoglobin. The reduced oxygen affinity of opossum hemoglobin may be compensated for by other physiological factors such as a reduced phosphate effect.