Replacement of valine by tryptophan or tyrosine at position $alpha$96 of the $alpha$ chain ($alpha$96Val), located in the ${alpha}_1{eta}_2$ subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the $alpha$96 position. The characteristic of aromatic amino acid substitution at the $alpha$96 of hemoglobin has been further investigated by producing double mutant r Hb ($alpha$42Tyr$ ightarrow$ Phe, $alpha$96Val$ ightarrow$Trp). r Hb ($alpha$42Tyr$ ightarrow$Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between $alpha$42Tyr and $eta$99Asp in the ${alpha}_1{eta}_2$ subunit interface of deoxy Hb A. The second mutation, $alpha$96Val$ ightarrow$Trp, may compensate the functional defects of r Hb ($alpha$42Tyr$ ightarrow$Phe), if the stability due to the introduction of trypophan at the $alpha$96 position is strong enough to overcome the defect of r Hb ($alpha$42Tyr$ ightarrow$Phe). Double mutant r Hb ($alpha$42Tyr$ ightarrow$Phe, $alpha$96Val$ ightarrow$Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb ($alpha$42Tyr$ ightarrow$Phe). $^1$H NMR spectroscopic data of r Hb ($alpha$42Tyr$ ightarrow$Phe, $alpha$96Val$ ightarrow$Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between $alpha$42Tyr and $eta$99Asp is essential for the novel oxygen binding properties of deoxy Hb ($alpha$96Val$ ightarrow$Trp).