Ligand migration in the apolar tunnel of cerebratulus lacteus mini-hemoglobin

ALESSANDRA PESCE; MARCO NARDINI; SYLVIA DEWILDE; LUCIANA CAPECE; MARCELO A. MARTI; SONIA CONGIA; MALLORY D. SALTER; GEORGE D. BLOUIN; DARIO A. ESTRIN; PAOLO ASCENZI; LUC MOENS; MARTINO BOLOGNESI; JOHN S. OLSON

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2010

0021-9258

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 Xe causes loss of diffraction in wild-type (wt) CerHbO2 crystals, but Leu86(G12)Ala CerHbO2, which has an increased tunnel volume, stably accommodates two discrete Xe atoms: one adjacent to Leu86(G12) and another near Ala55(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. Addition of 10-15 atm of Xe to solutions of wt CerHbCO and L86A CerHbCO causes 2 to 3-fold increases in the fraction of geminate ligand recombination indicating that the bound Xe 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 to 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 Xe to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln44(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 Gln44(E7) conformer is thermodynamically favored.