Determination of ligand pathways in globins: apolar tunnels versus polar gates.

Although molecular dynamics simulations suggest multiple interior pathways for O(2) entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.

Ligand migration in the apolar tunnel of Cerebratulus lacteus mini-hemoglobin.

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray 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.

The apolar channel in Cerebratulus lacteus hemoglobin is the route for O2 entry and exit.

The major pathway for O2 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 (kO2) rate coefficients, but the changes are not systematic. More significantly, the fractions (Fgem approximately 0.05-0.19) and rates (kgem approximately 50-100 micros(-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 kO2 to decrease roughly 4-fold; 2) Fgem for CO to increase from approximately 0.05 to 0.45; and 3) kgem to decrease from approximately 80 to approximately 9 micros(-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.