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Acta Biochimica et Biophysica Sinica Advance Access originally published online on June 8, 2009
Acta Biochimica et Biophysica Sinica 2009 41(7):603-617; doi:10.1093/abbs/gmp048
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Functional and structural alterations induced by copper in xanthine oxidase

Mahnaz Hadizadeh1, Ezzatollah Keyhani2, Jacqueline Keyhani2,* and Cyrus Khodadadi1

1 Institute of Biochemistry and Biophysics, University of Tehran, PO Box 13145-1384, 13145 Tehran, Iran
2 Laboratory of Life Sciences, Saadat Abade, Sarve Sharghi 58, 19979 Tehran, Iran

* Correspondence address. Tel: +98-21-66956974; E-mail: keyhanie{at}ibb.ut.ac.ir or keyhanius2002{at}yahoo.com


  Abstract

Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Here, copper's ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cu2+ concentrations for various periods of time. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. Results showed that Cu2+ either stimulated or inhibited XO activity, depending on metal concentration and pre-incubation length, the latter also determining the inhibition type. Cu2+–XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and {alpha}-helical and β-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu2+ binding sites in the vicinity of the enzyme's reactive centers. Data indicated that Cu2+ binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. Stimulation was attributed to transient stabilization of XO optimal conformation. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties.

Keywords    xanthine oxidase; enzyme activity; spectroscopy; metal–enzyme complex; copper

Received: January 23, 2009; Accepted: April 3, 2009
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