Carbon monoxide binding to the ferrous chains of [Mn,Fe(II)] hybrid hemoglobins: pH dependence of the chain affinity constants associated with specific hemoglobin ligation pathways.

In mixed-metal [Mn,Fe] hybrid hemoglobins (Hb), the two chains of a single type, alpha or beta, are substituted with manganese protoporphyrin IX, which does not bind CO in either the Mn(II) or Mn(III) valency states. Thus, CO binding by the two ferrous subunits of a hybrid with Mn of either valency represents a simplified two-step Hb ligation process in which ligands bind to a single-chain type. Considering the [Mn(II),Fe(II)] hybrids, which are deoxy T-state analogues, at pH 6.6 both types bind CO with low affinity (alpha-Fe, 0.38 mmHg; beta-Fe, 0.71 mmHg) and noncooperatively (Hill coefficient n = 1). At elevated pH, both exhibit an increase in affinity (Bohr effect) and strong cooperativity, with the alpha-Fe hybrid having a higher degree of cooperativity (n approximately equal to 1.6) than beta-Fe (approximately equal to 1.3) at pH 9.0. The CO association constants for the Hb ligation routes in which the first two ligands bind to the same chain type are obtained from these measurements, and their pH dependence provides estimates of the proton release at each step. Through studies of CO on- and off-rates, the [Mn(III),Fe(II)] hybrids are used to obtain the pH dependence of the association constants for binding the fourth CO to the individual Hb chains.(ABSTRACT TRUNCATED AT 250 WORDS)