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Water penetration and binding to ferric myoglobin
W Cao1, J F Christian, P M Champion
1Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115, USA.
Biochemistry
|May 9, 2001
Summary
Flash photolysis reveals how water enters horse heart metmyoglobin after nitric oxide (NO) dissociates. His-64 displacement is key for small molecule entry and exit, impacting NO rebinding.
Area of Science:
- Biochemistry
- Photochemistry
- Protein dynamics
Background:
- Metmyoglobin (Mb(3+)) binds nitric oxide (NO) and water.
- Understanding ligand kinetics in heme proteins is crucial.
Purpose of the Study:
- To investigate the kinetics of water entry into horse heart metmyoglobin after NO photodissociation.
- To elucidate the role of His-64 in gating small molecule access to the heme pocket.
Main Methods:
- Flash photolysis of Mb(3+)NO.
- Kinetic measurements of water binding and NO rebinding.
- Study of His-64 mutants (H64L, H64G).
Main Results:
- Determined the rate constant for water binding (k(w) ≈ 5.7 x 10^6 s^-1) and dissociation constant (60 mM) at 293 K.
- Identified an Arrhenius barrier height (H(w) = 42 ± 3 kJ/mol) for water binding, similar to CO escape.
- Observed a 3-order of magnitude enhancement in NO rebinding for H64L and H64G mutants.
Conclusions:
- Water entry and small molecule exchange in metmyoglobin are gated by His-64 displacement.
- Hydrogen bonding involving His-64 stabilizes water binding, preventing NO scavenging by ferric heme proteins.