Deep Red Blood Cell Proteome Defines the Band 3 N-Terminus Interactome as a Regulator of Hypoxic Adaptation via BLVRB-Dependent S -Nitroso Transfer

Abstract Red blood cells (RBCs) have long been regarded as passive oxygen carriers, yet growing evidence reveals a complex, dynamic proteome independent of de novo gene expression. Here, we define the erythrocyte as an oxygen-responsive system organized around a Band 3 (SLC4A1)–centered metabolon. Using deep proteomics of ultra-pure RBCs and cross-linking interactomics, we identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that binds the N-terminal cytosolic domain under normoxia and dissociates under hypoxia, when band 3-deoxyhemoglobin interactions increase threefold. This reversible interaction forms an oxygen-sensitive switch coupling structural, redox, and metabolic remodeling. In humanized mice, truncation of the Band 3 N-terminus disrupted glycolytic activation, reduced 2,3-bisphosphoglycerate synthesis, and impaired exercise tolerance despite preserved cardiopulmonary function, establishing the physiological relevance of this module. Population-scale proteome quantitative trait locus (pQTL) analyses revealed coordinated variation of SLC4A1 and BLVRB abundance but minimal association of biliverdin levels with BLVRB genotype, suggesting alternative functions beyond heme catabolism. Mechanistically, BLVRB Cys109 acts as a nitric oxide (NO) relay, trans-nitrosating glycolytic enzymes such as GAPDH at active site Cys152, transiently inhibiting glycolysis. This S-nitrosation–mediated feedback mirrors conserved mechanisms in plants, where GAPDH-SNO redirects carbon flow toward the Calvin–Benson cycle under nitrosative stress, revealing an evolutionary convergence in gas-responsive metabolic control. Collectively, our findings define a Band 3–BLVRB–hemoglobin axis that links oxygen sensing, NO signaling, and redox homeostasis, providing a unifying model for how an anucleate cell achieves environmental adaptability through reversible protein–protein interactions and post-translational chemistry. Graphic abstract Issaian et al. define the most comprehensive proteome of ultra-pure human red blood cells (3,775 proteins) and map the O₂-dependent interactome, revealing a Band 3–BLVRB–hemoglobin module that links oxygen sensing to metabolic remodeling via reversible inhibitory S-nitrosation of GAPDH C152. In plants this redirects carbon toward photosynthesis, illustrating a conserved NO-dependent metabolic reprogramming mechanism across oxygen-regulated systems. Highlights Deep proteomics defines a complete, contamination-free RBC proteome (3,775 proteins) Cross-linking proteomics maps an oxygen-sensitive Band 3-centered interactome O2-dependent BLVRB–Band 3 binding regulates metabolism via S-nitrosation of GAPDH Band 3 N-terminus is required for hypoxic remodeling and exercise tolerance in vivo Full Text Availability The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.