The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

The major pathway for O-2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k'(O2)) and dissociation (k(O2)) rate coefficients, but the changes are not systematic. More significantly, the fractions (F-gem approximate to 0.05-0.19) and rates (k(gem) approximate to 50-100 mu s(-1)) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k'(O2) and k(O2) to decrease roughly 4-fold; 2) F-gem for CO to increase from similar to 0.05 to 0.45; and 3) k(gem) to decrease from similar to 80 to similar to 9 mu s(-1), as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway.