The neuronal hemoglobin from Cerebratulus lacteus (CerHb) has an O2 affinity (P50 ≈ 1 muM) similar to mammalian myoglobin (Mb), which are both optimized for oxygen storage and release. Mb has a histidine at the E7 helical position, which provides a strong, stabilizing hydrogen bond to bound O2 and a leucine and valine at the B10 and E11 positions, respectively. In contrast, CerHb has three potential hydrogen bonding donors, tyrosine at B10 and glutamine at E7, called the YQ motif, and an unusual Thr at the E11 position. Invertebrate hemoglobins displaying the YQ motif typically have much higher oxygen affinities (P50 ≤ 0.1 muM) and significantly lower rates of O2 dissociation (≤5 s-1) than Mbs. Using mutagenesis and IR spectroscopy, we have been able to show that the beta-hydroxyl of ThrE11 accepts a proton from the TyrB10 side chain, causing the non-bonded electrons of the phenoxyl group to point toward bound O2. The resultant partial negative charge destabilizes the FeO2 complex, causing the high oxygen dissociation rate constant, kO2, and moderate affinity of the wild-type protein.