A study of the K+-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side

A series of ferric and ferrous derivatives of wild-type ascorbate peroxidas e (APX) and of an engineered K+-site mutant of APX that has had its potassi um cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4 degrees C. Wild-typ e ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five -coordinate high-spin heme ligated on the proximal side by a histidine at p H 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-h istidine). The K+-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compa red with spectra of the wild-type APX. The electronic absorption and MCD sp ectra of the engineered K+-site APX mutant are essentially identical to tho se of cytochrome bg a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K+-site mutant of APX has undergone a conform ational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational chan ge is the result of mutagenesis of the protein to remove the K+-binding sit e which is located similar to 8 Angstrom from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.