Title

The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site

Authors

Authors

B. A. Dow; N. Sukumar; J. O. Matos; M. Choi; A. Schulte; S. A. Tatulian;V. L. Davidson

Comments

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Abbreviated Journal Title

Arch. Biochem. Biophys.

Keywords

Amicyanin; Cupredoxin; Hydrogen bond; Protein structure; TRANSFER PROTEIN COMPLEX; METHYLAMINE DEHYDROGENASE; PARACOCCUS-DENITRIFICANS; X-RAY; DIRECTED MUTAGENESIS; CRYSTAL-STRUCTURE; SPECTROSCOPIC PROPERTIES; STRUCTURAL DETERMINANTS; ANGSTROM RESOLUTION; MOLECULAR-GRAPHICS; Biochemistry & Molecular Biology; Biophysics

Abstract

The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it is separated by 10.1 angstrom. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pK(a) value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the beta-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence. (C) 2014 Elsevier Inc. All rights reserved.

Journal Title

Archives of Biochemistry and Biophysics

Volume

550

Publication Date

1-1-2014

Document Type

Article

Language

English

First Page

20

Last Page

27

WOS Identifier

WOS:000336780300003

ISSN

0003-9861

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