Paramagnetic probes of the domain structure of histidine-rich glycoprotein

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Abstract
The interaction of Cu2+ and Fe3+-mesoporphyrin with histidine-rich glycoprotein (HRG) from rabbit serum was examined spectroscopically. The first equivalent of Cu2+ binds to HRG producting a type II electron paramagetic resonance (EPR) spectrum with g.dblvert. = 2.25, gm = 2.05, A.dblvert. = 0.019 cm-1 (180 G), and superhyperfine along gm. These spectral parameters suggest moderately covalent coordination of Cu2+ to the protein by nitrogens. With increasing Cu2+ the superhyperfine disappears; however, the g and A values change only marginally. The increase in EPR signal amplitude throughout the addition of 1-15 equiv of Cu2+ is linear and thereafter maximizes, suggesting 18-22 equiv are bound. In contrast, changes in the circular dichroism spectrum at 280 nm appear sigmoidal and can be interpreted as the binding of Cu2+ to two structurally distinct regions of the protein. Evidence for two structurally distinct binding domains is found by comparing EPR spectra of Cu2+ complexes of HRG with spectra from complexes of two of its major proteolysis products (peptides). After binding 1 equiv of Cu2+, both the 30-kDa histidine-rich peptide and the native protein exhibit identical spectra including the pronounced superhyperfine. In contrast, the spectrum of the histidine-normal 45-kDa peptide with 1 equiv of Cu2+ bound lacks superhyperfine and parallels closely that of the native protein with 20 equiv bound. Finally, Fe3+-mesoporphyrin binds to HRG exhibiting both high-spin (g = 6.05) and low-spin (gz = 2.94, gy = 2.25, gx = 1.50) EPR resonances, and the latter imply bis(histidine) coordination. The amplitude of the low-spin gz resonance decreases throughout the addition of 5-20 equiv of Cu2+, and the Fe3+-mesoporphyrin high-spin responance increases and becomes rhombic at 10 equiv. The nitrogen superhyperfine is clearly visible even with Fe3+-mesoporphyrin bound. Taken together, these data are consistent with Cu2+ binding to two structurally distinct metal binding domains of HRG and a mostly sequential saturation of these domains. With increasing Cu2+ bound, Cu2+ coordination appears to progress from predominately histidine in the 30-kDa domain to aspartic acid and glutamic acid in the 45-kDa domain.

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