Flash Photolytic Studies of Carbon Monoxide Binding to the Ferrous Chains of [Mn(II),Fe(II)] Hybrid Hemoglobins: Kinetic Mechanism for the Early Stages of Hemoglobin Ligation

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn-(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the a chains or β chains. At elevated pH, CO binding to the a chains produces a larger degree of T to R conversion than binding to the β chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both α- and β-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.