Abstract
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.
Highlights
The pathways for ligand binding in truncated 2/2 Hbs appear to be distinct from the E7 gate in the classic 3-on-3 ␣-helical globins (19 –22)
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
Blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both kO2 and kO2 to decrease roughly 4-fold; 2) Fgem for CO to increase from ϳ0.05 to 0.45; and 3) kgem to decrease from ϳ80 to ϳ9 s؊1, as ligands become trapped in the channel
Summary
There are no large concomitant decreases in the O2 dissociation rate coefficient, and as a result, there are significant ϳ10-fold decreases in O2 affinity for the larger Phe-44 and Trp-44 CerHb mutants (Table 1) These effects for the larger 44(E7) amino acids appear to be due to direct side chain interactions with the bound ligand and not alterations in the speed of entry and escape. O2 and CO Binding to Position 55(E18) Mutants—Ala-55(E18) is located at the exit and entry point of the apolar channel between the E and H helices To see if this entrance could be blocked, Ala-55(E18) was replaced with Val, Leu, Phe, and Trp, with the premise that the barrier to ligand uptake would increase with increasing size of the amino acid side chain. Equilibrium, and geminate constants for O2 and CO binding to position 55(E18) mutants of CerHb at pH 7, 20 °C
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