Characterization of the spleen green hemeprotein with magnetic and natural circular dichroism spectroscopy: positive evidence for a myeloperoxidase-type active site

Abstract

The green hemeprotein purified from bovine spleen has been characterized with magnetic and natural circular dichroism (MCD and CD) spectroscopy for the first time. The enzyme derivatives studied include the native high-spin ferric form and its high-spin chloride and low-spin cyanide and nitrite complexes, the ligand-free high-spin ferrous form and its low-spin CO adduct, and Compounds II (ferryl iron species) and III (dioxygen adduct). All these enzyme states exhibit MCD spectra that are considerably different from the spectra of analogous complexes of normal heme iron. In particular, the following distinctions have been observed. The sign of the derivative-shaped MCD bands of the high-spin ferric and Compound II forms in the Soret (380-500 nm) region and of the ferrous low-spin and Compound III forms in both the Soret and visible (500-700 nm) regions are opposite to and, except for the high-spin ferric form, are less symmetric than those seen for normal heme iron systems. MCD intensities in the Soret region for the high-spin ferrous and low-spin ferric derivatives are noticeably smaller than those of normal heme proteins by a factor of up to ten. Prominent MCD bands are seen around 450 and 630 nm for the green hemeprotein derivatives; these features are considerably red-shifted (30-50 nm) relative to the analogous transitions observed for normal heme proteins. In contrast to the aforementioned spectral differences, the MCD and CD spectra of the spleen green hemeprotein derivatives are essentially identical to those previously reported for several derivatives of another spectroscopically anomalous heme-type enzyme, myeloperoxidase. This provides strong evidence that the two enzymes have identical prosthetic groups and endogenous axial ligands coordinated to the central iron. The novel MCD features of the green proteins, taken together with previously reported spectroscopic results, are most consistent with the presence of a chlorin-type prosthetic group in both proteins. In addition, the CD spectral similarities suggest that the two green proteins have nearly identical active-site environments.