To investigate conformational information of a low oxygen affinity recombinant hemoglobin (rHb) containing $96Val{ ightarrow}Trp$ mutation at the ${alpha}96$ position, we ave produced rHb (${alpha}96Val{ ightarrow}Phe$) and rHb (${alpha}96Val{ ightarrow}Tyr$), using the Escherichia coli expression system and site-directed mutagenesis. The oxygen affinity of rHb (${alpha}96Val{ ightarrow}Phe$) is similar to that of human normal adult hemoglobin (Hb A). However, the oxygen affinity of rHb (${alpha}96Val{ ightarrow}Tyr$) showed much lower oxygen affinity than Hb A which is similar to that of rHb (${alpha}96Val{ ightarrow}Tyr$), providing an opportunity as a potential candidate for a hemoglobin-based blood substitute. Both rHb (${alpha}96Val{ ightarrow}Phe$) and rHb (${alpha}96Val{ ightarrow}Tyr)$ showed high cooperativity in oxygen binding. IH-NMR spectroscopy shows that both rHb (${alpha}96Val{ ightarrow}Phe$) and rHb (${alpha}96Val{ ightarrow}Tyr$) have very similar tertiary structure around the heme pockets and uaternary structure in the ${alpha}_1/{eta}_2$ subunit interface ompared to Hb A. The low oxygen affinity of rHb (${alpha}96Val{ ightarrow}Tyr$) has been suggested to be due to a hydrogen bond caused by an extra hydroxyl group not present in rHb (${alpha}96Val{ ightarrow}Phe$). However, investigation of the carbonmonoxy form of rHb (${alpha}96Val{ ightarrow}Phe$) and (${alpha}96Val{ ightarrow}Try$) in the presence of inositol hexaphosphate at low temperature suggests that low oxygen affinity of (${alpha}96Val{ ightarrow}Try$) may arise from a mechanism different to that of rHb (${alpha}96Val{ ightarrow}Trp$).