Apo-myoglobin (apo-Mb) and apo-A71GMb were successfully reconstituted with Fe-III(salophen) (1) (salophen = N,N'-bis(salicylidene)-1,2-phenilenediamine), Fe-III(3,3'-Me2-salophen) (2), and Fe-III (5,5'- t-Bu-2-salophen) (3). The crystal structure of 2-apo-A71GMb shows the tight binding of the complex in the Mb cavity, while in wild-type apo-Mb it is highly disordered due to the steric repulsion with Ala71. Furthermore, the structure of 2-apo-A71GMb suggests a possible accommodation of a small substrate in the cavity. In fact, the cyanide association rate constant of 2-apo-A71GMb is 216-fold larger compared to that of 2-apo-Mb. These results provide us principles for the noncovaient fixation of synthetic metal cofactors at the desired positions in protein matrixes.