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Investigations of the alkaline and acid transitions of umecyanin, a stellacyanin from horseradish roots

Lookup NU author(s): Professor Christopher Dennison, Anne Lawler

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Abstract

The effect of pH on Cu(I) and Cu(II) umecyanin (UCu), a phytocyanin obtained from horseradish roots, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pKa value of ∼9.5-9.8 is observed for the alkaline transition in UCu(II), and this leads to a slightly altered active site structure, as indicated by the changes in the paramagnetic 1H NMR spectrum. Electrochemical studies show that the pKa value for this transition in UCu(I) is 9.9. The alkaline transition is caused by the deprotonation of a surface lysine residue, with Lys96 being the most likely candidate. The isotropically shifted resonances in the 1H NMR spectrum of UCu(II) also shift upon lowering the pH (pKa 5.8), and this can be assigned to the protonation of the surface (noncoordinating) His65 residue. This histidine titrates in UCu(I) with a pKa of 6.3. The reduction potential of the protein in this range is also dependent on pH, and pKa values matching those from NMR, for the two oxidation states of the protein, are obtained. There is no evidence for either of the active site histidines (His44 and His90) titrating in UCu(I) in the pH range studied (down to pH 3.7). Also highlighted in these studies are the remarkable active site similarities between umecyanin and the other phytocyanins which possess an axial Gln ligand.


Publication metadata

Author(s): Lawler AT; Dennison C

Publication type: Article

Publication status: Published

Journal: Biochemistry

Year: 2001

Volume: 40

Issue: 10

Pages: 3158-3166

ISSN (print): 0006-2960

ISSN (electronic): 1520-510X

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/bi002020j

DOI: 10.1021/bi002020j

PubMed id: 11258931


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