Kinetic spectroscopy of heme hydration and ligand binding in myoglobin and isolated hemoglobin chains: an optical window into heme pocket water dynamics

Abstract

The entry of a water molecule into the distal heme pocket of pentacoordinate heme proteins such as myoglobin and the α,β chains of hemoglobin can be detected by time-resolved spectroscopy in the heme visible bands after photolysis of the CO complex. Reviewing the evidence from spectrokinetic studies of Mb variants, we find that this optical method measures the occupancy of non(heme)coordinated water in the distal pocket, n_w, with high fidelity. This evidence further suggests that perturbation of the kinetic barrier presented by distal pocket water is often the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Water entry into the heme pockets of isolated hemoglobin subunits was detected by optical methods. Internal hydration is higher in the native α chains than in the β chains, in agreement with previous crystallographic results for the subunits within Hb tetramers. The kinetic parameters obtained from modeling of the water entry and ligand rebinding in Mb mutants and native Hb chains are consistent with an inverse dependence of the bimolecular association rate constant on the water occupancy factor. This correlation suggests that water and ligand mutually exclude one another from the distal pockets of both types of hemoglobin chains and myoglobin.