The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by xray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO 2 crystals, but Leu-86(G12)Ala CerHbO 2 , which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10 -15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (<25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.
Although molecular dynamics (MD)7 simulations suggest multiple interior pathways for O 2 entry into and exit from globins, most experiments with mammalian myoglobins (Mbs) and hemoglobins (Hbs) suggest a well defined single pathway involving a short channel between the heme propionates and the heme iron atom that is gated by the distal E7 histidine (1). To search for and define an interior ligand migration trajectory, we chose to use the neuronal mini-hemoglobin from Cerebratulus lacteus as a model globin system to examine its long apolar tunnel that leads from the distal portion of the heme pocket to an exit point between the C-terminal regions of the E and H helices of the tertiary fold, a pathway that is roughly 180°opposite the E7 channel and appears to be a major route for ligand entry (2). This model globin provides a well defined system to examine both experimentally and theoretically the effects of xenon binding, mutagenesis, and conformational heterogeneity on the competition between movement through the E7 gate versus an internal apolar pathway.Nerve tissue Hbs occur in both vertebrates and invertebrates (3). Among these, the nerve Hb from the nemertean worm C. lacteus (CerHb) is the smallest functional globin known, being composed of 109 amino acids instead of the ϳ140 -160 residues typical of most monomeric globins (4, 5). Analysis of the three-dimensional structure of CerHb has shown a markedly edited 3-over-3-globin fold with deletion of