Recombinant Hemoglobin rHb0.1 with Cross-Linked Alpha Subunits Preferentially Crystallizes in the β₄ Oligomeric...

Hemoglobin rHb0.1 is a recombinant version of the red blood cell transport protein carrying cross-linked α-subunits (V1M and G15A) and β-subunit mutations (V1M, G16A, and H116I), which is used as a construct to develop recombinant hemoglobin-based oxygen carriers (rHBOCs). Spectroscopic studies revealed the altered stability of rHb0.1 compared to heterotetrameric (α₂β₂) HbA. Heme dissociation kinetic analysis revealed nearly 2-fold faster rate constants (k _slow and k _fast) for rHb0.1 compared to HbA, with such differences necessitating structural studies for a greater insight into the variabilities. Here, we report the first crystal structure of the oxygen-bound β₄-rHb0.1 at 2.0 Å resolution, obtained from crystallization conditions designed for α₂β₂ rHb0.1, reflecting preferential crystallization of a preexisting β₄ population in solution. The overall quaternary architecture in the oxy-bound state closely resembles ferric, deoxygenated, and carbomonoxy β₄ HbA and liganded α₂β₂ R-state HbA. Unlike ferric β₄-HbA, the oxy-bound β₄-rHb0.1 structure prevents the formation of a disulfide bond between Cys112-(G14) residues of β1/β4 and β2/β3 subunits. A detailed comparison of the quaternary structures of oxy-bound (O₂-β₄) and ferric β₄ (PDB: 6FQF) showed minimal conformational changes upon ligand binding to the β₄ tetramer (RMSD across all 146 pairs: 0.714), demonstrating the remarkable structural conservation of β₄ tetramers in different redox states. Lower expression of the di-α subunit compared to the β-subunit in solution seems to have triggered β₄ homotetramer formation, as revealed by the crystal structure, with the H116I mutation in the β-subunit promoting this homotetramerization through extensive hydrophobic β-β interactions, offering insights into hemoglobin assembly, stability, and functional divergence from HbA.