Characterization of Cu2+ and Fe3+ -mesoporphyrin complexes with histidine-rich glycoprotein: evidence for Cu2+ -Fe3+ -mesoporphyrin interaction.

One equivalent of Fe3+ -mesoporphyrin (heme) is coordinated by two axial histidine ligands to a preferred site on histidine-rich glycoprotein (HRG). This study shows that titration of this stochiometric heme.HRG complex with 0-20 equivalents of Cu2+ produces a series of pronounced spectral changes indicative of multiple, sequential alterations of the heme environment. A monotonic low- to high-spin heme transition characterized by a decrease in resonance amplitude at g = 2.99, an increase at g = 6.0, and an increase in absorptivity at 620 nm is induced with the addition of the first 10 Cu2+ equivalents. Furthermore, optical absorption and circular dichroism spectra exhibit isosbestic and isodichroic points throughout the addition of the first 8 and 12 equivalents, respectively. The isosbestic points imply a transition between two optically well defined axial heme coordinations, and the isodichroic points suggest that these axial coordinations also represent two distinct protein conformations. A second isosbestic is formed during the addition of 14-20 equivalents of Cu2+, again suggesting well-defined coordinations; however, changes in the EPR spectra over this range are more complex. Whereas the amount of low-spin (g = 2.99) heme.HRG complex continues to decrease with the addition of 10-20 Cu2+ equivalents, the amount of the high-spin (g = 6.0) complex reaches a maximum near 14 equivalents and decreases markedly thereafter. Of potentially greater significance is the appearance of signals at g = 9.3 (maximum), 7.7 (maximum), 4.8 (crossover), and 1.61 (minimum) after addition of 10 or more Cu2+ equivalents. Some of these signals are similar to those exhibited by cardiac cytochrome c oxidase upon reduction and reoxidation. Thus, even without the addition of exogenous reductants and oxygen, the interaction of Cu2+ with the stoichiometric heme.HRG complex may produce structural features similar to those found in a mechanistically important but poorly understood form of cardiac cytochrome c oxidase.