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Spectroscopic studies revealed the altered stability of rHb0.1 compared to heterotetrameric (\u03b12\u03b22) HbA. Heme dissociation kinetic analysis revealed nearly 2-fold faster rate constants (kslow and kfast) 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 \u03b24-rHb0.1 at 2.0 \u00c5 resolution, obtained from crystallization conditions designed for \u03b12\u03b22 rHb0.1, reflecting preferential crystallization of a preexisting \u03b24 population in solution. The overall quaternary architecture in the oxy-bound state closely resembles ferric, deoxygenated, and carbomonoxy \u03b24 HbA and liganded \u03b12\u03b22 R-state HbA. Unlike ferric \u03b24-HbA, the oxy-bound \u03b24-rHb0.1 structure prevents the formation of a disulfide bond between Cys112(G14) residues of \u03b21\/\u03b24 and \u03b22\/\u03b23 subunits. A detailed comparison of the quaternary structures of oxy-bound (O2-\u03b24) and ferric \u03b24 (PDB: 6FQF) showed minimal conformational changes upon ligand binding to the \u03b24 tetramer (RMSD across all 146 pairs: 0.714), demonstrating the remarkable structural conservation of \u03b24 tetramers in different redox states. Lower expression of the di-\u03b1 subunit compared to the \u03b2-subunit in solution seems to have triggered \u03b24 homotetramer formation, as revealed by the crystal structure, with the H116I mutation in the \u03b2-subunit promoting this homotetramerization through extensive hydrophobic \u03b2-\u03b2 interactions, offering insights into hemoglobin assembly, stability, and functional divergence from HbA.<\/jats:p>","DOI":"10.1021\/acsomega.5c11376","type":"journal-article","created":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T13:24:31Z","timestamp":1778160271000},"page":"27896-27912","source":"Crossref","is-referenced-by-count":0,"title":["Recombinant Hemoglobin rHb0.1 with Cross-Linked Alpha Subunits Preferentially Crystallizes in the \u03b24 Oligomeric State, Potentially Driven by a \u03b2G18(H116I) Mutation"],"prefix":"10.1021","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-0254-2350","authenticated-orcid":true,"given":"Kajal","family":"Yadav","sequence":"first","affiliation":[{"name":"University of Delhi , , South Campus, Benito Juarez Marg , ,","place":["New Delhi, India, 110021"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3382-1839","authenticated-orcid":true,"given":"Shalja","family":"Verma","sequence":"additional","affiliation":[{"name":"IIT Roorkee , , , ,","place":["Roorkee, Uttarakhand, India, 247667"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8433-9209","authenticated-orcid":true,"given":"Alo","family":"Nag","sequence":"additional","affiliation":[{"name":"University of Delhi , , South Campus, Benito Juarez Marg , ,","place":["New Delhi, India, 110021"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2128-7736","authenticated-orcid":true,"given":"Pravindra","family":"Kumar","sequence":"additional","affiliation":[{"name":"IIT Roorkee , , , ,","place":["Roorkee, Uttarakhand, India, 247667"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8960-2096","authenticated-orcid":true,"given":"Suman","family":"Kundu","sequence":"additional","affiliation":[{"name":"University of Delhi , , South Campus, Benito Juarez Marg , ,","place":["New Delhi, India, 110021"]},{"name":"Birla Institute of Technology and Science Pilani , , K K Birla Goa Campus , , ,","place":["Zuarinagar, Goa, India, 403726"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,5,7]]},"reference":[{"issue":"3","key":"2026081816065994100_cit1","doi-asserted-by":"publisher","first-page":"a011858","DOI":"10.1101\/cshperspect.a011858","article-title":"Hemoglobin variants: Biochemical properties and clinical correlates","volume":"3","author":"Thom","year":"2013","journal-title":"Cold Spring Harbor Perspect. 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