Cooperative nitric oxide binding by manganese hemoglobin. Implications for the role of steric control in hemoglobin ligation.

The reaction of NO with manganoglobin, manganous porphyrin-substituted hemoglobin (MnHb), shows close correspondence to the reaction of CO with hemoglobin (Hb) in all features examined. All measurements are consistent with cooperative binding of NO by MnHb, associated with a T + R conformation change in the tetramer. An important conclusion based on a comparison of rates of NO ligation by Hb and MnHb is that steric control of access to the metal center by a ligand cannot be responsible for the change in ligation rate caused by the hemoglobin T -+ R transformation. The apparent second order rate constant for the (NO + MnHb) combination reaction is about 36 m~-l s-’ (pH 7 and 2O”C), compared with about 120 mu-’ s-l for the (CO + Hb) reaction. Both reactions are autocatalytic and liganding is associated with similar changes in reactivity with bromothymol blue (faster with MnHb than with MnHbNO) and j?-mercuribenxoate (slower with MnHb than with MnHbNO). Binding of 8-hydroxyl-1,3,6-pyrenetrisulfonate to MnHb is stronger than to MnHbNO, although the difference is less than that between Hb and HbCO. The dissociation of NO from MnHb in the presence of dithionite is slow (0.005 s-l), suggesting a static dissociation constant of the order of lo-’ M. As with HbCO, MnHbNO is photosensitive with a similarly high quantum yield. In both cases, the products of partial photodissociation react 20 to 30 times faster with their respective ligands than the products of complete photodissociation. Spectrophotometric evidence of an R + T conformational transformation upon NO photodissociation is also presented. The tetramer-dimer dissociation constants of metM&b, MnHbNO, and MnHb are 1 X lo-‘, 1 x lo-’ M, and too small to measure accurately with the ultracentrifuge. Dimeric MnHb, like dimeric Hb, reacts 20 to 30 times faster with ligand than the tetramer does. The reduction of Mn3+ porphyrin of Mn”%Ib by dithionite and the oxidation of MnHb by ferricyanide are both markedly biphasic reactions. Analogy with the corresponding reaction of Hb suggests that chain differences are responsible.