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 peroxidase (APX) and of an engineered K(+)-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4 degrees C. Wild-type 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 pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K(+)-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K(+)-site APX mutant are essentially identical to those of cytochrome b5, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K(+)-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K(+)-binding site which is located approximately 8 A 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.