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Comparative Analysis of Oxy-Hemoglobin and Aquomet-Hemoglobin by Hydrogen/Deuterium Exchange Mass Spectrometry

  • Research Article
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Journal of The American Society for 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.

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Acknowledgments

The authors acknowledge support for this work by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, and the Canada Research Chairs Program. They thank Dr. Jiangjiang Liu for her help during preparation of the protein samples that were used for this study.

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  1. Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada

    Modupeola A. Sowole & Lars Konermann

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  1. Modupeola A. Sowole
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  2. Lars Konermann
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Correspondence to Lars Konermann.

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Sowole, M.A., Konermann, L. Comparative Analysis of Oxy-Hemoglobin and Aquomet-Hemoglobin by Hydrogen/Deuterium Exchange Mass Spectrometry. J. Am. Soc. Mass Spectrom. 24, 997–1005 (2013). https://doi.org/10.1007/s13361-013-0647-4

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