title: Comparative analysis of oxy-hemoglobin and aquomet-hemoglobin by hydrogen/deuterium exchange mass spectrometry.
abstract: The function of hemoglobin (Hb) as oxygen transporter is mediated by reversible O2 binding to Fe(2+) heme in each of the α and β subunits. X-ray crystallography revealed different subunit arrangements in oxy-Hb and deoxy-Hb. The deoxy state is stabilized by additional contacts, causing a rigidification that results in strong protection against hydrogen/deuterium exchange (HDX). Aquomet-Hb is a dysfunctional degradation product with four water-bound Fe(3+) centers. Heme release from aquomet-Hb is relatively facile, triggering oxidative damage of membrane lipids. Aquomet-Hb crystallizes in virtually the same conformation as oxy-Hb. Hence, it is commonly implied that the solution-phase properties of aquomet-Hb should resemble those of the oxy state. This work compares the structural dynamics of oxy-Hb and aquomet-Hb by HDX mass spectrometry (MS). It is found that the aquomet state exhibits a solution-phase structure that is significantly more dynamic, as manifested by elevated HDX levels. These enhanced dynamics affect the aquomet α and β subunits in a different fashion. The latter undergoes global destabilization, whereas the former shows elevated HDX levels only in the heme binding region. It is proposed that these enhanced dynamics play a role in facilitating heme release from aquomet-Hb. Our findings should be of particular interest to the MS community because oxy-Hb and aquomet-Hb serve as widely used test analytes for probing the relationship between biomolecular structure in solution and in the gas phase. We are not aware of any prior comparative HDX/MS experiments on oxy-Hb and aquomet-Hb.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Modupeola A
authors.initials: MA
authors.lastname: Sowole
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D041961:Deuterium Exchange Measurement / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008706:Methemoglobin / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010108:Oxyhemoglobins / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D021122:Protein Subunits
chemical_list: D010108:Oxyhemoglobins; D010446:Peptide Fragments; D021122:Protein Subunits; C024963:aquomethemoglobin; D008706:Methemoglobin