Most proteins associate into multimeric complexes with specific architectures, which often have functional properties like cooperative ligand binding, allosteric regulation, or the capacity to perform mechanical work. We have no detailed knowledge of how any multimer and its functions arose during historical evolution. Here we use ancestral protein reconstruction and biophysical assays to dissect the evolutionary origins of vertebrate hemoglobin (Hb), a heterotetramer of paralogous α and β subunits, which mediates oxygen transport and exchange by binding cooperatively to oxygen. We show that modern Hb evolved from an ancient monomer, characterize the historical “missing-link” dimeric form through which the modern tetramer evolved, and establish that two historical substitutions at this dimer’s protein surface were sufficient to confer tetramerization. Acquisition of this quaternary association dramatically alters the oxygen-binding function and confers cooperativity. These observations reveal that evolution can produce new molecular complexes and confer new functional properties via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.

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