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

ABSTRACT: 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.