The red blood cell proteome and interactome identify a Band 3-BLVRB axis regulating hypoxic metabolic adaptation.

Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultrapure human RBCs, we generated the deepest contamination-free proteome to date (3775 proteins) and mapped the oxygen-dependent interactome. These data sets revealed an oxygen-responsive metabolon centered on the Band 3 (SLC4A1) N terminus. We identified biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that dissociates under hypoxia, coincident with increased Band 3-deoxyhemoglobin contacts. This reversible assembly functions as an oxygen-sensitive switch coordinating redox and glycolytic remodeling. Humanized mice lacking Band 3 N-terminal segments exhibited impaired oxygen-dependent regulation of BLVRB binding to Band 3, impaired hypoxic activation of glycolysis, reduced 2,3-bisphosphoglycerate synthesis, and diminished exercise tolerance, demonstrating physiological relevance. Population-scale cis-protein quantitative trait loci for SLC4A1 and BLVRB suggest functions beyond canonical heme catabolism. Mechanistically, in vitro biochemical analyses suggest that hemoglobin β, Band 3, and BLVRB can undergo S-nitrosation and may participate in transnitrosation reactions with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, whose modification at C152 inhibits enzymatic activity in vitro. Collectively, these findings define a Band 3-BLVRB axis that integrates oxygen-dependent protein interactions with thiol-based redox chemistry, providing a framework for understanding how an anucleate cell achieves metabolic adaptability through reversible protein-protein interactions and posttranslational modification. These findings suggest that the perturbation of the Band 3-BLVRB axis may influence oxygen delivery and metabolic flexibility during hypoxic stress, with potential relevance to high-altitude adaptation, exercise physiology, and cardiopulmonary disease.