Highly Asymmetric Interactions between Globin Chains during Hemoglobin Assembly Revealed by Electrospray Ionization Mass Spectrometry

Publisher Website
Google Scholar
Abstract
Dynamics of bovine hemoglobin assembly was investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. Intensities of ionic signals corresponding to various protein species (tetramers, dimers, heme-deficient dimers, as well as apo- and holo-monomers) were used to estimate relative fractions of these species in solution as a function of pH. The fraction of folded protein for each observed species was estimated based on charge-state distributions of corresponding ionic species in the mass spectra. The cumulative numbers (averaged across the entire protein population) were in good agreement with circular dichroism data at the Soret band and in the far-UV region, respectively. The mass spectral data confirm that hemoglobin dissociation involves a step where heme is first lost from the β-chain of the αβ-dimer to form a heme-deficient dimeric species. This dimer dissociates further to produce a holo-α-chain and an apo-β-chain. The former is tightly folded into a comparatively compact structure at neutral pH, while the latter always exhibits significant backbone disorder. Acidification of the protein solution to pH 4 leads to partial heme dissociation and significant increase of the backbone flexibility in the α-chains as well. Complete dissociation of the heme from the α-chains at a pH below 4 coincides with the total disappearance of the dimeric and tetrameric hemoglobin species from the mass spectra. The experimental data provide strong evidence that binding of a partially unstructured apo-β-chain to a tightly folded holo-α-chain to form a heme-deficient dimer is the initial step of hemoglobin assembly. Such binding locks the β-chain in a highly ordered conformation, which allows for an efficient heme acquisition, followed by docking of two hemoglobin dimers to form a tetrameric form of the protein. The asymmetry of the roles of the two chains in the assembly process is surprising, given a rather high sequence homology (ca. 43%) and highlights functional importance of intrinsic protein disorder. The study also demonstrates a tremendous potential of mass spectrometry as an analytical tool capable of elucidating protein interaction mechanisms in highly heterogeneous systems.