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 H-1 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 H-1 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.