ISSN:
1432-1327
Keywords:
Key words Peroxidase
;
Magnetic circular dichroism
;
Ascorbate peroxidase
;
Heme-containing enzyme
;
Site-directed mutagenesis
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s007750050290
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