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.
Cheek
J
J
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
Mandelman
D
D
Poulos
T L
TL
Dawson
J H
JH
D060387:Ascorbate Peroxidases; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004583:Electrons; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008280:Magnetics; D008969:Molecular Sequence Data; D009154:Mutation; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods
D006418:Heme; D007501:Iron; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D011188:Potassium