Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities.
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