A non-coordinated water molecule within the apolar pocket plays an essential role in regulating ligand binding in both myoglobin and hemoglobin. The occupancy of this non-coordinated water molecule in the distal pocket of these heme proteins has been determined with high-fidelity using a kinetic spectral marker, a shift in the 560 nm deoxy absorption peak occurring ~500 ns after photodissociation of CO, under a variety of conditions using Time Resolved Absorption Spectroscopy. This work extends the technique to examine Mb mutants with different axial ligands  (H93C and H93Y) with the goal of being able to determine the distal pocket water occupancy in a larger class of heme proteins, including the P450s.  The spectral signature for water occupancy is assigned using a combination of Soret and visible band kinetics and double mutants (H64L/H93C and H64L/H93Y) known to remove this water from the heme pocket.

Measuring the entry of non-coordinated water in the distal pocket of myoglobin proximal ligand mutants

243rd National Meeting (2012)

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