ABSTRACT
The functional properties of the Arg residue at the E10 helical position in myoglobin and hemoglobin lacking the highly conserved His residue at the E7 position have received considerable interest as to the structure-function relationship of the oxygen-binding hemoproteins, because Arg E10 in such proteins has been shown to play similar roles to those His E7 plays in ordinary proteins. One of the components of hemoglobin from the larval hemolyph of Tokunagayusurika akamusi is also a naturally occurring E7 genetic variant with Ile E7 and Arg E10. This study demonstrated, for the first time, that the positively charged, elongated, and flexible side-chain of Arg E10 in T.akamusi hemoglobin contributes to stabilization of the coordination of biologically relevant Cl(-) to heme iron. Determination of the dynamics of the Cl(-) binding to T. akamusi ferric hemoglobin involving paramagnetic 1H-NMR indicated that the Cl(-) affinity increases with decreasing pH as a result of the fact that the binding rate increases with decreasing pH, whereas the dissociation rate is almost completely independent of pH. The pH-dependent character of the Cl(-) binding rate correlated well with the ionization state of heme peripheral side-chain propionate groups, which was clearly manifested in the pH-dependent shift changes of heme methyl proton signals, suggesting that negative charges of heme propionate groups constitute a kinetic barrier for Cl(-) entry into the heme pocket. These findings provide an insight into the pH-dependent ligand binding properties of T. akamusi hemoglobin.
