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 O-2 dissociation and ultra-high O-2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O-2 dissociation (k(O2) = 200 - 600 s(-1)) and moderate O-2 affinity (K-O2 approximate to 1 muM(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, k(O2) decreases 1000-fold and K-O2 increases 130-fold at pH 7.0, 20 degreesC. 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 --> Val CerHbO(2) at 1.70 Angstrom resolution is almost identical to that of the wildtype protein ( root mean square deviation of 0.12 Angstrom). The dramatic functional and spectral effects of the Thr-E11 --> 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 O-2 affinity, a decrease of the rate coefficient for O-2 dissociation, a 40 cm(-1) decrease in nu(CO) of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.