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

P.F Hollenberg,L.P Hager,W.E Blumberg,J Peisach

doi:10.1016/s0021-9258(19)85568-0

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

Highlights

The electron paramagnetic resonance spectra of chlo- Chloroperoxidase,isolatedfrom the mold Caldariomyces roperoxidase, a heme protein isolated from the mold fumago, is a heme proteinwith a molecular weight of approx
A t liquid helium temperatures, the EPR spectrum of native chloroperoxidase prepared at pH1.5 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 increasingthe pH above 1.5 causes a pH-dependent spin transitionfrom 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

Summary

Since the hemeiron is thought tobe intimatelv involved in

E P R Spectra of Chloroperoxidase the catalytic mechanism of peroxidases, a study of the elec- these studies hadspecific activities greater than 2,000 units/mg of tronic propertiesof the hemeiron, the immediate environment proteinin the standardchlorination assay andexhibited A4,,:,/A2m, of the hemeiron, and theeffect of substrate binding on these properties using EPR may provide vital information about the mechanism of action of this enzyme. EPR Spectra-The samples used for EPR experiments were prepared by diluting 0.3 ml of concentrated chloroperoxidase (approximately 80 mg/ml) with 0.4 ml of the appropriate ligand-containing on studies by Stern and Peisach [20], Collman and Sorrell [21],and Chang andDolphin [22] on cytochromeP-450 model compounds, Hansonand co-workers [19] suggest that the anomaloushyperspectrum of the reduced CO complex of cytochrome P-450 is duetothe presence of a mercaptide sulfur as an axial ligand to theiron. Upon cooling to 77 K the spectrum native nor the urea-denatured formofschloroperoxidase con- changes markedly, with the Soret moving to 424 nm, peaks tain a free sulfhydryl group They concluded that appearing at 543 and 582 nm, and the concomitant disappearthe axial ligand of chloroperoxidase wasnot a sulfhydryl group ance of the peaks at 516 and 652 nm. 400 to 388 nm) while the addition ofC1- results in a shift in the opposite direction (400 to 412 nm)

CT nrn Native"

Hyper Soret C T '

DISCUSSION

The resulting computer high field g values aregiven in Table