Abstract
We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation ( Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254-1259 ). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.
Highlights
Leaving the sixth coordination position free to bind small molecule ligands
Earlier kinetic studies of myoglobin found that the observed rate of CO binding increases as the pH is lowered below neutral (18 –23), increasing in sperm whale Mb3 by more than a factor of 10 on going from pH 7 to 3 [23]. (In contrast to the changes observed at low pH, Lambright et al [24] measured CO binding kinetics in human myoglobin from pH 6.5 to 10 and found that the rates were independent of both pH and salt concentration.) Most of the increase in observed ligand binding rate constant occurs at pH values below 4 and has been attributed previously to protonation of the proximal histidine, which disrupts the iron-His93 bond and converts deoxy-Mb to a tetracoordinated form [20, 23, 25]
We investigate in the present work the variation of heme pocket hydration with pH to: 1) determine the extent to which modulation of this kinetic barrier to CO entry contributes to the changes in the CO bimolecular rebinding rate constant induced by pH, and 2) investigate the role that protonation of the distal histidine in particular plays in modulating the kinetics of heme hydration and ligand association
Summary
We found that mutations of the distal histidine altered the intrinsic rates of both water exit and entry, rates that together determined water occupancy in the pocket Changes in this water occupancy were found to be the dominant factor modulating the observed bimolecular association rate constant for CO in the mutants studied. The resulting loss of water occupancy in this scenario would explain much of the faster rate of diffusive ligand rebinding to the heme iron atom that has been attributed to the low pH conformation (28 –31) The implications of this finding for the larger perturbation of ligand binding kinetics observed previously at pH Ͻ 4 are discussed below
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