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 |
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Authors: | Jennifer Cheek David Mandelman T. L. Poulos J. H. Dawson |
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Affiliation: | (1) Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA e-mail: dawson@psc.sc.edu, Fax: +1-803-7779521, US;(2) Department Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA e-mail: poulos@uci.edu, Fax: +1-949-8243280, US;(3) School of Medicine, University of South Carolina, Columbia, SC 29208, USA, US |
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Abstract: | 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??°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 b 5, 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 ~8?Å 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. |
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Keywords: | Peroxidase Magnetic circular dichroism Ascorbate peroxidase Heme-containing enzyme Site-directed mutagenesis |
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