Water Penetration and Binding to Ferric Myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb3+NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb3+ (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme...

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Bibliographic Details
Published in:Biochemistry (Easton) 2001-05, Vol.40 (19), p.5728-5737
Main Authors: Cao, Wenxiang, Christian, James F, Champion, Paul M, Rosca, Florin, Sage, J. Timothy
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Animals
Binding Sites - genetics
Heme - chemistry
Heme - metabolism
Histidine - genetics
Horses
Hydrogen Bonding
Kinetics
Leucine - genetics
Ligands
Metmyoglobin - chemistry
Metmyoglobin - genetics
Metmyoglobin - metabolism
Models, Chemical
Nitric Oxide - chemistry
Nitric Oxide - metabolism
Water - chemistry
Water - metabolism
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Summary:Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb3+NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb3+ (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb3+H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k w ≈ 5.7 × 106 s-1 for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H w = 42 ± 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb2+CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi010067e