Metal-Enzyme Frameworks: Role of Metal Ions in Promoting Enzyme Self-Assembly on α-Zirconium(IV) Phosphate Nanoplates

Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamics of protein binding to negatively charged alpha-Zr(IV) phosphate (alpha-ZrP). This model is tested here using glucose oxidase (GO) and met-hemoglobin (Hb) and several cations (Zr(IV), Cr(III), Au(III), ARM), Ca(II), Zn(II), Ni(II), Na(I), and H(I)). The binding constant of GO with alpha-ZrP was increased similar to 380-fold by the addition of either 1 mM Zr(IV) or 1 mM Ca(II), and affinities followed the trend Zr(IV) similar or equal to Ca(II) > Cr(III) > Mg(II) >> H(I) > Na(I). Binding studies could not be conducted with Au(III), Zn(II), Cu(II), and Ni(II)), as these precipitated both proteins. Zr(IV) increased Hb binding constant to alpha-ZrP by 43-fold, and affinity enhancements followed the trend Zr(IV) > H(I) > Mg(II) > Na(I) > Ca(II) > Cr(III). Zeta potential studies clearly showed metal ion binding to alpha-ZrP and affinities followed the trend, Zr(IV) >> Cr(III) > Zn(II) > Ni(II) > Mg(II) > Ca(II) > Au(III) > Na(I) > H(I). Electron microscopy showed highly ordered structures of protein/metal/alpha-ZrP intercalates on micrometer length scales, and protein intercalation was also confirmed by powder X-ray diffraction. Specific activities of GO/Zr(IV)/alpha-ZrP and Hb/Zr(IV)/alpha-ZrP ternary complexes were 2.0 x 10(-3) and 6.5 x 10(-4) M-1 s(-1), respectively. While activities of all GO/cation/alpha-ZrP samples were comparable, those of Hb/cation/alpha-ZrP followed the trend Mg(II) > Na(I) > H(I) > Cr(III) > Ca(II) similar or equal to Zr(IV). Metal ions enhanced protein binding by orders of magnitude, as predicted by the ICPB model, and binding enhancements depended on charge as well as the phosphophilicity/oxophilicity of the cation.