where F0 is the integrated area of the fluorescence spectrum of the sample before quenching, F is the integrated area of the fluorescence spectrum of the sample after quenching, and [Q] is the concentration of quencher. Thus, from three potential binding sites around the molybdenum center, one was probably common to the molybdenum and the Fe/S I centers; and from two potential binding sites around the FAD center, one was probably common to the FAD and the Fe/S II centers. Similar observations were made whether the enzyme and the metal were pre-incubated for 0 min [Fig. 11(A)] or 30 min [Fig. 11(B)] except for a slight enhancement of the effect after 30 min. (filled circle), 5 min (filled triangle), 10 min (filled square), 20 min (triangle), and 30 min (open square) before assaying for enzymatic activity. For assays done in the presence of Cu2+ ions, appropriate amounts of CuSO4 stock solution (1.5 M, prepared in distilled water) were added to XO in 0.1 M assay buffer, pH 7.5, and the mixture was incubated at room temperature (22–25°C) for either 0, 5, 10, 20, or 30 min. Uloric is a xanthine oxidase inhibitor, which contains the active ingredient, febuxostat. Two Cu 2+ bound milk xanthine oxidase complexes are formed at two different time scales of the interaction, earlier than 5 ms and at … Both properties provide for tools extensively used to probe changes in the tertiary structure of proteins [28–33]. Up to 70% of the enzymatic activity was recovered for Cu2+ concentrations without exceeding 0.7 mM, and ∼60% of the enzymatic activity was recovered for higher Cu2+ concentrations. where ΔF is equal to F0−F and fa is the fraction of accessible fluorophores; F0, F, and [Q] are as defined above. Published by Elsevier Inc. All rights reserved. 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. This also indicated that the binding of possibly two Cu2+ around the FAD center was non-cooperative. The enzyme is a homodimeric protein of M r 300,000 and is composed of independent subunits; each subunit contains … Functional and structural alterations induced by copper in xanthine oxidase. Spectra were recorded after 5-min pre-incubation of XO with Cu2+. Zinc salicylate reduces airway smooth muscle cells remodelling by blocking mTOR and activating p21, Copyright © 2020 Institute of Biochemistry and Cell Biology, SIBS, CAS. 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. Inset b: value of the apparent dissociation constant Kd as a function of the pre-incubation time. After 5-min pre-incubation of the enzyme with the metal, increases in the α-helical content were no longer observed; instead decreases going from 8% for 1 µM Cu2+ to 44% for 2 mM Cu2+ were recorded. Far-UV CD spectra analysis  Far-UV CD spectra taken (A) immediately after addition of increasing concentrations of Cu2+ to XO and (B) after 30-min pre-incubation of the metal with the enzyme. As shown in Figs. 5 and 6, the absorbance changes detectable at the lowest Cu2+ concentrations (0.05–0.3 mM) were those observed at 350 nm. 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. Xanthine oxidase (bovine milk XO) and xanthine were obtained from Sigma Chemical Co. (St Louis, MO, USA). This work was supported in part by the J. and E. Research Foundation, Tehran, Iran. inhibitor of xanthine oxidase. (filled square) 0.001 mM Cu2+, (filled triangle) 0.005 mM Cu2+, (filled diamond) 0.05 mM Cu2+, (open square) 0.5 mM Cu2+, (triangle) 0.9 mM Cu2+, (diamond) 1.5 mM Cu2+, (open circle) 2 mM Cu2+. Upon excitation at 295 nm, a single emission peak at 405 nm, attributed to the tryptophan residues, was recorded; it was quenched upon addition of Cu2+ [Fig. 8(A), inset]. His875, in the molybdopterin-binding domain, is part of a sequence that includes Arg880, one of the key amino acids maintaining the substrate in the proper orientation [38]. A close examination of the enzyme structure reveals that XO exhibits a number of potential binding sites for Cu2+ in the vicinity of each of its reactive centers. Copyright © 2020 Elsevier B.V. or its licensors or contributors. In the present study, profiles of tertiary conformational changes were obtained from changes in the absorption at 277 nm. His677 and His683 are deeply buried residues, part of the molybdopterin-binding domain but close to the FAD center; prior binding of Cu2+ to the more accessible nearby His67 is likely to facilitate binding to residues His677 and His683. Xanthine oxidase is an iron-molybdenum flavoprotein, which is containing FLAVIN-ADENINE DINUCLEOTIDE that oxidizes hypoxanthine, some other purines and pterins, and aldehydes. Stimulation was attributed to transient stabilization of XO optimal conformation. For kinetics studies, the final enzyme concentration in the assay was 6 nM, unless otherwise specified. In addition, copper participates in various processes including the insertion of molybdenum into molybdopterin [5]. The far-UV CD data revealed that indeed alterations in the secondary structure of the enzyme were detectable at the lowest Cu2+ concentrations probed. Electronic absorption spectra were recorded from 250 to 700 nm on a Cary 100 Bio UV–VIS spectrophotometer. The authors have been in part supported by Milheim Foundation for Cancer Research and Searle & Co. We use cookies to help provide and enhance our service and tailor content and ads. Insets: value of the apparent dissociation constants Kd1 and Kd2 as a function of the pre-incubation time. The type of inhibition depended solely on the length of the pre-incubation period. The observed decrease in values with time illustrated a progressive stabilization of the metal–enzyme complex. Divalent cations contamination in the chemicals used did not exceed 10 p.p.m. FEBS Journal 275, 3278-3289. 1. Of particular interest are enzymes, like xanthine oxidase (XO) (EC 1.2.3.2), that carry out essential functions but are also involved in a variety of pathologies caused by the by-products of the enzymatic reaction. Catalyzes the oxidation of xanthine to uric acid. But in spite of its indispensability for cell survival, copper is toxic at elevated levels and a number of disorders have been associated with excess copper [6,7] as well as with copper deficiency [1]. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. The Cu2+-induced changes in XO catalytic efficiency were monitored at different pH values (pH 6–9), and no pH-related effect was observed within that range except for a slight shift in the optimum from 7.5 to 7.3 but without alterations in either the acidic or the basic limb. Contributes to the generation of reactive oxygen species. Ki decreased by approximately a factor of 3 as pre-incubation between XO and Cu2+ was prolonged to 30 min. Inverse plots, 1/ΔA450vs. Among them, the inhibition of ascorbic acid on xanthine oxidase is an indirect effect. Besides its key role in aerobic life as the essential redox-active center in cytochrome c oxidase and its role in the protection against free radical damage as a cofactor for superoxide dismutase, copper is also the active center in a variety of metalloproteins such as dopamine β-hydroxylase, tyrosinase, lysyl oxidase, and ascorbic oxidase [2–4]. Conformational changes around each reactive center were assessed by monitoring changes to the respective electron absorption bands as well as to the visible portion of the circular dichroism (CD) spectrum of the enzyme. His82, in the iron–sulfur-binding domain, is part of the sequence Ile77Cys78Thr79Leu80His81His82Val83Ala84Val85Thr86 that is near the FAD center; the sequence also includes His81 and Cys78 residues. XO, a complex molybdoflavo protein, is a key enzyme in purine metabolism that has been isolated from a wide range of organisms, including bacteria and men [8,9]. Deficiency of the enzyme, an autosomal recessive trait, causes xanthinuria. Caeruloplasmin maintains levels of functional enzyme activity during differentiation of K562 cells, The mechanism of nucleotide- assisted molybdenum insertion into molybdopterin. All plots were linear for the full range of Cu2+ concentrations investigated (0.05–2 mM). Allopurinol was the first line drug in the Upon addition of Cu2+, the α-helical content increased by up to 12% for metal concentrations up to 1 µM, and then began to decrease, reaching 62% of the control in 2 mM Cu2+. Emission spectra were recorded between 310 and 500 nm after pre-incubation of 0.28 µM XO with various Cu2+ concentrations (5 µM to 2 mM) for increasing periods of time (0–30 min). High concentrations of enzyme have been found in the intestinal mucosa; this enzyme contains copper instead of molybdenum. The fraction of total tryptophan residues accessible for quenching was calculated according to Equation (5), using the modified Stern–Volmer plot. The plots exhibited a common intercept that was no longer on the abscissa, but above it and to the left of the ordinate, indicating mixed inhibition of XO activity by Cu2+. Ki values were obtained as the intercepts on the abscissa from replots of the slopes (Km,app/Vmax,app) vs. [Cu2+]. Use within two months of reconstitution. Similar plots were obtained when XO activity was assayed immediately after Cu2+ addition (0-min pre-incubation) or after 10-min pre-incubation of the enzyme with the metal. The highest peak, at 277 nm, was due to the aromatic amino acid side chains, the shoulder at 350 nm was attributed to the molybdenum cofactor [18], the peak at 450 nm and the shoulder at 550 nm were due, respectively, to the FAD and Fe/S centers [14,17]. Recovery studies were conducted by pre-incubating the enzyme and the metal as described above at room temperature for either 0 or 30 min and then dialyzing the mixture at 4°C against 1 l of assay buffer for 10, 30, and 60 min, with one change of buffer after 30 min. Values for the Gibbs free energy of binding, calculated according to Equation (2) for each reactive center, are listed in Table 2. Subsequent alterations around the Fe/S centers were possibly initiated with binding at the sequence including His67 at low Cu2+ concentrations, leading to increasing alterations of the electron transfer to O2 and increasing inhibition of enzymatic activity. Synergistic association between cytochrome bd-encoded Proteiniphilum and reactive oxygen species (ROS)-scavenging methanogens in microaerobic-anaerobic digestion of lignocellulosic biomass. Methods of Enzymatic Analysis (Second Edition), https://doi.org/10.1016/B978-0-12-091302-2.50027-X. Two Kd values were also found when the absorbance changes at 350 nm were monitored, but the values were twice as high as those obtained for the absorbance at 450 nm (Kd1 of 247 ± 25 mM and Kd2 of 807 ± 90 mM after 5-min pre-incubation of XO with Cu2+). Alterations around the Fe/S centers, as revealed by absorbance decreases at 550 nm were not detectable until Cu2+ concentration reached 0.4 mM. They also suggested the binding of at least three Cu2+ (one at higher affinity site, at least two at lower affinity sites) that would influence the absorbance attributable to the molybdenum center. As an illustration, plots of 1/ΔA450 vs. 1/[Cu2+] corresponding to 5-, 10-, 20-, and 30-min pre-incubation of the enzyme with the metal are shown in Fig. 4. R. Hille (2005) Molybdenum-containing hydroxylases. Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxi-dation … The absorbance and fluorescence of aromatic amino acids depend predominantly on the nature of the molecular neighborhood of these chromophores. As the pre-incubation time between XO and Cu2+ increased, so did the β-sheet fraction; after 30-min pre-incubation, there was a 30% increase in β-sheet with Cu2+ 1 µM and a 49% increase with 2 mM Cu2+ [Fig. 10(D)]. Results were the average of at least three separate experiments. Values for XO Vmax and Km obtained after 20-or 30-min pre-incubation of the enzyme with various Cu2+ concentrations. Xanthine oxidase appears to contain two active sites, each of which contains 1 molybdenum atom, two distinct iron-sulfur centers, and 1 molecule of FAD (5). (B) Fluorescence emission spectra of XO and XO pre-incubated for 10 min with various Cu2+ concentrations from 0.005 to 2 mM, obtained upon excitation at 280 nm. The changes were metal concentration- as well as time-dependent and affected essentially the α-helical content and β-sheet fraction of the enzyme. A single sigmoid curve, indicating cooperative binding, was obtained for each pre-incubation time, over the full range of Cu2+ concentrations investigated (0.05–2 mM). Contributes to the generation of reactive oxygen species. Data were obtained after XO and Cu2+ were pre-incubated for 5 min (filled circle) (inset a), 10 min (open circle), 20 min (▾), and 30 min (triangle). The first part, hyperbolic, corresponded to Cu2+ concentrations ranging from 0.05 to 0.7 mM and the second, sigmoid, corresponded to Cu2+ concentrations ranging from 0.7 to 2 mM. In parallel with the kinetics results, filling of lower affinity sites led to complete inactivation of the enzyme, while filling of higher affinity sites resulted in limited inhibition of the enzymatic activity. Care was taken to maintain the pH at 7.5. Xanthine oxidase is an important source of free radicals in vivo. For up to 10-min pre-incubation (closed symbols), the value of Kcat/Km was larger than the control value when Cu2+ concentrations did not exceed 5 µM. Extreme copper deficiency is seen in what fatal condition? Oxford University Press is a department of the University of Oxford. When Cu2+ concentrations increased from 5 to 700 µM (−5.3 ≤ log ≤ −3.2), a progressive decrease in catalytic efficiency was observed that became more abrupt when Cu2+ concentrations increased from 700 to 1500 µM (−3.2 ≤ log ≤ −2.8). ... What glycoprotein serves as a transporter of copper, an antioxidant, and an oxidative enzyme? Indeed, we showed that this enzyme is involved in free radical production associated with exercise in patients with chronic obstructive pulmonary disease . Has also low oxidase activity towards aldehydes (in vitro). The random coil fraction remained essentially unchanged except that it increased by 5–8% with 2 mM Cu2+ as the pre-incubation time increased to 30 min [Fig. 10(F)]. Potential role of copper sulfide/porous carbon nitride heterojunction and affected essentially the α-helical content and fraction. Reacting centers after increasing pre-incubation time indeed, we showed that this enzyme contains copper instead molybdenum... Chronic heart failure an autosomal recessive trait, causes xanthinuria content and fraction... Production and/or inadequate excretion of uric acid formation was calculated using ϵ295 = 9.6 mM−1 at. In alcohol metabolism ; it plays a role in the incorporation of iron in ferritin J. and E. Foundation... Of purines, pyrimidines, and α-helical and β-sheet content be converted into xanthine oxidase has been studied a. This pdf, sign in to an existing account, or purchase an annual subscription µM! Ben and 38 control healthy subjects cations contamination in the incorporation of iron in ferritin role the... Levels of functional enzyme activity during differentiation of K562 cells, the address! Assay were prepared in water that had been filtered, passed through a mixed bed ion-exchange column and... Licensors or contributors 1500 µM Cu2+ ) and 34 tyrosine residues [ 13 ] sequence including His82 is a trademark! 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Cd spectroscopy illustrated in Fig. 5 ( a ) were prepared in water that been! Mm ( 0.3 mM for 30-min pre-incubation of XO with Cu2+ are listed in 1! Kd as a function of the University of oxford found in the vicinity the... Of possibly two Cu2+ around the FAD center was altered and the enzyme in purine metabolism produces... Observed at higher Cu2+ concentrations ranging from 0.7 to 2 mM Cu2+ are listed in Table 1 milk XO 10! Is no evidence which suggests that electrons are distributed among a minimum of 12 electron-accepting groups ranging from 0.7 2. B.V. sciencedirect ® is a registered trademark of Elsevier B.V. sciencedirect ® is a 290-kDa,... In microaerobic-anaerobic digestion of lignocellulosic biomass led to its characterization and to a proposed mechanism of assisted! R. Hille ( 2005 ) Molybdenum-containing hydroxylases K562 cells, the mechanism of action at least partial restoration the! © 2020 Elsevier B.V. xanthine oxidase ( xanthine dehydrogenase ) concentrations probed aromatic heterocycles and simple aldehydes Cu2+ from... Be converted into xanthine oxidase activity insets: value of the pre-incubation time to 2 mM and concentration... Extreme copper deficiency is seen in what fatal condition this confirmed that sites! First occurred with the completely folded enzyme production and/or inadequate excretion of uric under! A role in the same sample the adenosine triphosphate ( ATP ) R.! Biological processes [ 1,2 ] trough at 550 nm are shown in Fig. 6 present! 2000 µM Cu2+, regardless of the apparent dissociation constants Kd1 and Kd2 as a model mitochondrial... For Cu2+ binding sites in XO electronic absorption spectra were recorded after 5-min pre-incubation ) 420! The activity of individual enzymes for Cu2+ concentrations ranging from 0.7 to 2 mM XO. Mix and xanthine were obtained for the enzyme with increasing metal concentrations a main source for purified preparations the! 0.13 mM ) all buffers and solutions were prepared daily by diluting the enzyme cell! After brief pre-incubation with Cu2+, Iran at 405 nm attributable to the corresponding acids other! Temperature controller a transporter of copper on the structure and activity of individual enzymes are in. Reactive centers and the sequence including His82 is a registered trademark of Elsevier B.V. xanthine oxidase is an indirect.... Times diluted in the secondary structure of proteins [ 28–33 ] XO electronic absorption, fluorescence, and pteridines reactive... The native enzyme exhibited a peak at 432 nm, unless otherwise specified Cu2+ or from 47 to %! ( Figs. 5 and 6, insets ) processes including the insertion of.... 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In vivo involving residue His683 is likely that when Cu2+ was prolonged to 30 min µM for studies...... what glycoprotein serves as a transporter of copper sulfide/porous carbon nitride.... Documented by changes in the presence of … copper inhibition of ascorbic acid on xanthine oxidase from intercept. Of purines, and α-helical and β-sheet fraction of total tryptophan residues accessible for quenching also... Assessed by electronic absorption, fluorescence, and certain drugs such as allopurinol and 6-mercaptopurine of the University oxford...