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.
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
Mallory D
MD
Salter
Karin
K
Nienhaus
G Ulrich
GU
Nienhaus
Sylvia
S
Dewilde
Luc
L
Moens
Alessandra
A
Pesce
Marco
M
Nardini
Martino
M
Bolognesi
John S
JS
Olson
D019943:Amino Acid Substitution; D000818:Animals; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D007448:Invertebrates / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D020125:Mutation, Missense; D017433:Protein Structure, Secondary / Q000235:genetics
D006454:Hemoglobins; D008024:Ligands