Measurement of distal histidine coordination equilibrium and kinetics in hexacoordinate hemoglobins.

The kinetics of ligand binding to hemoglobins has been measured for decades. Initially, these studies were confined to readily available pentacoordinate oxygen transport proteins like myoglobin, leghemoglobin, and red blood cell hemoglobin. Bimolecular ligand binding to these proteins is relatively simple, as ligand association is largely unimpeded at the heme iron. Although many techniques have been used to examine these reactions in the past, stopped-flow rapid mixing and flash photolysis are the most common ways to measure rate constants for ligand association and dissociation. Expression of recombinant proteins has allowed for examination of many newly discovered hemoglobins. The hexacoordinate hemoglobins are one such group of proteins that exhibit more complex binding kinetics than pentacoordinate hemoglobins due to reversible intramolecular coordination by a histidine side chain. Here, we describe methods for characterizing the kinetics of ligand binding to hexacoordinate hemoglobins with a focus on measurement of histidine coordination and exogenous ligand binding in both the ferrous and the ferric oxidation states.