Design and Synthesis of a Transition Metal Responsive Semisynthetic Myoglobin-Bearing Iminodiacetic Acid Moiety

Iminodiacetic acid appended myoglobins (IDAn-Mb, n = 1, 2) were synthesized by conventional reconstitution of chemically modified hemes with apomyoglobin. The metal responsive property of the obtained IDAn-Mb was studied by metal ion titration, pH titration, circular dichroism (CD) and ¹H NMR spectroscopies, and reduction with ascorbate. IDAn-Mb quantitatively bound various transition metal cations (Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Cu²⁺ but not Mg²⁺. The binding stoichiometry of IDA2-Mb was 1:1 for Co²⁺, Ni²⁺, Zn²⁺, and Cd²⁺ and 1:2 for Cu²⁺, whereas the stoichiometry of 1:1 was shown for IDA1-Mb to all transition metals (Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Cu²⁺). The acidic pK_a shift of the H₂O coordinated to the heme iron(III) was clearly observed upon the binding of transition metals, suggesting the microenvironmental change of the heme crevice. This was supported by the CD and ¹H NMR spectra of IDAn-Mb. The transition metal induced structural changes of IDAn-Mb were reflected in their redox behavior, i.e., the reduction rate of IDA2-Mb by ascorbate was enhanced 8-fold upon the Co²⁺ binding. The rate showed a good linear relationship with the shifted pK_a of the axial H₂O, indicating that the transition metal binding directly affects the electron acceptability of IDAn-Mb. These results demonstrated that iminodiacetic acid moieties can play a crucial role as a reporter molecule for design of a transition metal responsive semisynthetic protein.