Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 about 200–600 s-1) and moderate O2 affinity (KO2 about 1 microM-1) as a result of a third polar amino acid in its active site, Thr- E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 °C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 3 Val CerHbO2 at 1.70 Å resolution is almost identical to that of the wildtype protein (root mean square deviation of 0.12 Å). The dramatic functional and spectral effects of the Thr- E11 3 Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val “frees” the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm-1 decrease in CO of hemebound CO, and an increase in ligand migration toward more remote intermediate sites.