Origin of complexity in hemoglobin evolution

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.