Hemoglobin/O2Systems: Using Short-lived Intermediates for Mechanistic Discrimination

The kinetics of the reaction of hemoglobin with molecular oxygen, in which rapid mixing is followed by a very fast temperature jump, is numerically simulated. Four different mechanisms are considered. In two of them, oxygen reacts with theα-chains first, in the other two with theβ-chains first. Furthermore, either the third or the fourth measured (Adair) dissociation constant is composed of the product of a local dissociation constant and an allosteric interconversion constant. We explore whether these alternative mechanisms may be distinguished experimentally. We show that reaction steps not resolvable by rapid mixing can be resolved by chemical relaxation at appropriate points in time. Discrimination under experimental conditions is possible at higher oxygen concentrations (above 100 μM), but high resolution in time and concentration amplitude are required.