DESIGN AND SYNTHESIS OF A TRANSITION-METAL RESPONSIVE SEMISYNTHETIC MYOGLOBIN-BEARING IMINODIACETIC ACID MOIETY

Iminodiacetic acid appended myoglobins (IDAn-Mb, n = 1, 2) were synthe sized by conventional reconstitution of chemically modified hemes with apomyoglobin. The metal responsive property of the obtained IDAn-Mb w as studied by metal ion titration, pH titration, circular dichroism (C D) and H-1 NMR spectroscopies, and reduction with ascorbate. IDAn-Mb q uantitatively bound various transition metal cations (Co2+, Ni2+, Zn2, Cd2+, and Cu2+) but not Mg2+. The binding stoichiometry of IDA2-Mb w as 1:1 for Co2+, Ni2+, Zn2+, and Cd2+ and 1:2 for Cu2+, whereas the st oichiometry 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 coord inated to the heme iron(III) was clearly observed upon the binding of transition metals, suggesting the microenvironmental change of the hem e 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 refl ected 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, ind icating that the transition metal binding directly affects the electro n acceptability of IDAn-Mb. These results demonstrated that iminodiace tic acid moieties can play a crucial role as a reporter molecule for d esign of a transition metal responsive semisynthetic protein.