Ligand Migration in the Apolar Tunnel of Cerebratulus lacteus Mini-Hemoglobin.

ALESSANDRA PESCE; MARCO NARDINI; SYLVIA DEWILDE; LUCIANA CAPECE; MARCELO A. MARTI; S COGNIA; MD SALTER; CG BLOUIN; DARIO A. ESTRIN; PAOLO ASCENCZI; LUC MOENS; MARTINO BOLOGNESI; JOHN S OLSON

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2011 p. 5347 - 5358

0021-9258

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) hasbeen examined by X-ray crystallography, ligandbinding kinetics, and molecular dynamicsimulations. The addition of 10 atm of Xe causesloss of diffraction in wild-type (wt) CerHbO2crystals, but Leu86(G12)Ala CerHbO2, which hasan increased tunnel volume, stably accommodatestwo discrete Xe atoms: one adjacent to Leu86(G12)and another near Ala55(E18). Molecular dynamicssimulations of ligand migration in wt CerHb showa low energy pathway through the apolar tunnelwhen Leu or Ala, but not Phe or Trp, is present atthe 86(G12) position. Addition of 10-15 atm of Xeto solutions of wt CerHbCO and L86A CerHbCOcauses 2 to 3-fold increases in the fraction ofgeminate ligand recombination indicating that thebound Xe blocks CO escape. This idea wasconfirmed by L86F and L86W mutations, whichcause even larger increases in the fraction ofgeminate CO rebinding, 2 to 5-fold decreases in thebimolecular rate constants for ligand entry, andlarge increases in the computed energy barriers forligand movement through the apolar tunnel. Boththe addition of Xe to the L86A mutant andoxidation of wt CerHb heme iron cause theappearance of an out Gln44(E7) conformer, inwhich the amide side chain points out toward thesolvent and appears to lower the barrier for ligandescape through the E7 gate. However, the observedkinetics suggest little entry and escape (! 25%)through the E7 pathway, presumably because thein Gln44(E7) conformer is thermodynamicallyfavored.