Hemoglobin II (HbII) from the clam Lucina pectinata is a dimeric protein composed of 151 amino acids with high oxygen affinity. The structural organization in the heme pocket involving residues: Tyr30 (B10), Gln65 (E7), Phe69 (E11), and Phe44 (CD1) are associated with the O2 affinity. Recombinant HbII (rHbII) and the B10Phe mutant were cloned into the pCRT7/NT-TOPO vector, and expressed in Escherichia coli BL21 cells. The expression was performed with 0.4 mM and 1.0 mM IPTG for induction at 30ºC, 30 µg/mL hemin chloride was added to supplement the TB medium without glucose. UV-Vis spectra, size-exclusion chromatography on a Superose (12 10/300 GL) column and SDS-PAGE were used to monitor the presence of the protein. rHbII and HbIITyrB10Phe were purified by strong anion exchanger column. Nanosecond flash photolysis shows that rHbII and native HbII have similar oxygen binding properties, kon = 3 x 106 M-1s-1, koff = 0.3 s-1 and that rHbII is a good model of HbII. Kinetic studies show that the mutation of B10Phe leads to an increase of the dissociation rate of oxygen by one order of magnitude and the association rate increases by the same order kon= 4.65 x107 M-1s-1 and koff =5 s-1. These changes demonstrate the crucial role of the B10 residue in (1) the stabilization of the iron oxygen binding possibly through a strong hydrogen bond with the dipolar Fe-O2 bond; (2) the entry of the ligand from the solvent to the heme pocket.