title: Proton magnetic resonance study of the influence of chemical modification, mutation, quaternary state, and ligation state on dynamic stability of the heme pocket in hemoglobin as reflected in the exchange of the proximal histidyl ring labile proton.
abstract: Proton nuclear magnetic resonance spectroscopy has been utilized to investigate the rates of exchange with deuterium of the proximal histidyl ring protons in a series of chemically modified and mutated forms of Hb A. Differences in rates of exchange are related to differences in the stability of the deformed or partially unfolded intermediates from which exchange with bulk solvent takes place. Each modified/mutated Hb exhibited kinetic subunit heterogeneity in the reduced ferrous state, with the alpha subunit exhibiting faster exchange than the beta subunit. Modification or mutation resulted in significant increases in the His F8 ring NH exchange rates primarily for the affected subunit and only if the modification/mutation occurs at the allosterically important alpha 1 beta 2 subunit interface. Moreover, this enhancement in exchange rate is observed primarily in that quaternary state of the modified/mutated Hb in which the modified/substituted residue makes the intersubunit contact. This confirms the importance of allosteric constraints in determining the dynamic properties of the heme pocket. Using modified or mutated Hbs that can switch between the alternate quaternary states within a given ligation state or ligate within a given quaternary state, we show that the major portion of the enhanced exchange rate in R-state oxy Hb relative to T-state deoxy Hb originates from the quaternary switch rather than from ligation. However, solely ligation effects are not negligible. The exchange rates of the His F8 ring labile protons increase dramatically upon oxidizing the iron to the ferric state, and both the subunit kinetic heterogeneity and the allosteric sensitivity to the quaternary state are essentially abolished.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, University of California, Davis 95616.
authors.forename: K H
authors.initials: KH
authors.lastname: Han
authors.forename: G N
authors.initials: GN
authors.lastname: La Mar
authors.forename: K
authors.initials: K
authors.lastname: Nagai
mesh_terms: D000495:Allosteric Site; D002384:Catalysis; D003903:Deuterium; D004735:Energy Transfer; D006418:Heme; D006441:Hemoglobin A / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010084:Oxidation-Reduction; D011522:Protons
chemical_list: D008024:Ligands; D011522:Protons; D006418:Heme; D006639:Histidine; D006441:Hemoglobin A; D003903:Deuterium