An electron paramagnetic resonance study of the high and low spin forms of chloroperoxidase.

Abstract

The electron paramagnetic resonance spectra of chloroperoxidase, a heme protein isolated from the mold Caldariomyces fumago, and its complexes with a variety of substrates and inhibitors have been investigated. At liquid helium temperatures, the EPR spectrum of native chloroperoxidase prepared at pH 1.5 is essentially that of high spin ferric heme iron, while frozen solutions of the enzyme at pH 5 exhibit a low spin spectrum. At pH 3.0, both the high and low spin forms of chloroperoxidase are observed. These results indicate that chloroperoxidase exists as a pH-dependent equilibrium mixture of high and low spin forms at low temperature, and that increasing the pH above 1.5 causes a pH-dependent spin transition from a high spin form having g values of 7.64,4.29, and 1.78 to a low spin form having g values near 2.61, 2.26, and 1.83. The binding of such ligands as Br-, I-, and F- to the enzyme results in a shift in the equilibrium that favors the high spin form while the addition of such ligands as cyanide, azide, imidazole, and 2-mercaptoethanol shifts the equilibrium almost completely towards low spin forms. The addition of chloride has no major effect on the high spin-low spin equilibrium. Analysis of the EPR spectrum for the high spin form of native chloroperoxidase shows that it exhibits an extremely large rhombic distortion (-20%). The addition of such ligands as F-, C1-, Br-, and I- not only alters the high spin-low spin equilibrium but also alters the rhombicity, indicating that the binding of halide ligands to the enzyme perturbs the symmetry of the heme as well. An analysis of the EPR spectrum of the low spin forms of chloroperoxidase indicates that the electronic environment of the heme iron bears closer resemblance to cytochrome P-450 than to various nitrogen-, oxygen-, and carbon-ligated low spin forms of myoglobin, hemoglobin, or horseradish peroxidase. However, the electronic environment of