Functional and structural alterations induced by copper in xanthine oxidase.
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 Cu(2+) 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 Cu(2+) either stimulated or inhibited XO activity, depending on metal concentration and pre-incubation length, the latter also determining the inhibition type. Cu(2+)-XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and alpha-helical and beta-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu(2+) binding sites in the vicinity of the enzyme's reactive centers. Data indicated that Cu(2+) 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.
Hadizadeh
Mahnaz
M
Institute of Biochemistry and Biophysics, University of Tehran, 13145 Tehran, Iran.
Keyhani
Ezzatollah
E
Keyhani
Jacqueline
J
Khodadadi
Cyrus
C
D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002417:Cattle; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000378:metabolism*
D003300:Copper; D014969:Xanthine Oxidase