SummaryThe 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..

Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by x-ray crystallography. Here we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well defined, non-coordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring non-specific hydration of the active sites of heme proteins.

determining the stability and rigidity of proteins, shifting the pKa values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers. Detecting these internal water molecules is sometimes obscured in x-ray crystallography due to positional disorder. We have developed a spectrokinetic assay that accurately detects the presence of a non-coordinated water molecule in the distal heme pocket of myoglobin and in a series of distal pocket mutants, including many where this water molecule is positionally disordered. We also have shown that this water plays a major role in determining the observed bimolecular recombination rate constant. We show that 1) this water molecule modulates the ligand binding dynamics of a series of H64, L29 and V68 mutants; 2) it plays the major role in the observed pH dependence of the CO recombination kinetics between pH 4 and 7 with the protonation of the distal histidine acting as a switch to change water occupancy; and 3) it may also modulate ligand binding dynamics in isolated hemoglobin chains, with the occupancy being larger in the alpha chains. Accurately measuring water occupancy in heme proteins answers crucial questions about water in apolar or slightly polar protein cavities and clarifies the role internal water molecules play in modulating protein function.