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Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root

Lookup NU author(s): Dr Katsuko Sato, Dr Dr MD Harrison Harrison, Professor Christopher Dennison

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Abstract

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His2Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic 1H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pKa of ∼10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the Nε2H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction. © 2005 American Chemical Society.


Publication metadata

Author(s): Delfino I, Sato K, Harrison MD, Andolfi L, Bizzarri AR, Dennison C, Cannistraro S

Publication type: Article

Publication status: Published

Journal: Biochemistry

Year: 2005

Volume: 44

Issue: 49

Pages: 16090-16097

Print publication date: 13/12/2005

ISSN (print): 0006-2960

ISSN (electronic): 1520-4995

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/bi051702+

DOI: 10.1021/bi051702+

PubMed id: 16331969


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