Reductive enzymes xanthine oxidase

Fig. 5. Multiple phases in the reduction of xanthine oxidase by xanthine at pH 8.2. Intensities of the Rapid (circles) and Slow (triangles) molybdenum EPR signals expressed as electron/mole enzyme (i-e. per 2 atom Mo) are plotted as a function of time. Note the changes in the time scale. Rapid freezing was used for reaction times (at 22°) up to 1 sec. and manual mixing for longer times (at 25°) enzyme concentrations (immediately after mixing) were 0.09 mM and 0.13 mM respectively. The enzyme had Activity/A45o 125 corresponding to 63% of active enzyme and 20 mole xanthine/mole enzyme was used. (Data from ref. 67.)...

The "toxicity" of O2 is exerted primarily by its penetration to important sites, where it is converted into further, more reactive oxygen species. When a reaction between O2 and carbohydrates was observed, this was nearly exclusively the case when complex biomaterials, for instance cartilage [231] or body fluids, for instance joint (synovial) fluid [232], were used for the experiments. Such complex materials do always contain at least traces of transition metals. This was convincingly demonstrated by Carlin and Djursater [233]. These authors have shown that there is no reduction of the viscosity of a given HA solution when solely the enzyme xanthine oxidase is used as source of 02. In contrast, a marked depolymerization of HA occurs in the presence of ferritin-... 

During the reduction of xanthine oxidase the impulse polarographic wave displays two maxima at potentials 0.08 and -0.01 The position of the first maximum coincides with the enzyme redox potential. In a solution of denatured xanthine oxidase, the first maximum is absent. Accordingly, the view of the authors is that xanthine oxidase in this potential region is reduced in the native state. As for the other maximum, it is possibly associated with the reduction of sulfur-containing groups. 

Scheme 1. Possible oxidation-reduction reactions between reducing and oxidizing substrate molecules (R and O respectively) and the molybdenum (M), flavin (F) and iron (I) of xanthine oxidase. The enzyme molecule is represented by the circle and arrows indicate transfer of reducing equivalents...

Finally, in the case of inhibitory substrate analogues such as allo-xanthine, strong evidence has recently been presented that these bind to molybdenum in reduced xanthine oxidase (33). If the enzyme is reduced with xanthine, then treated anaerobically with alloxanthine and finally exposed to air, catalytic activity is lost. Though flavin and iron in the final product are in the oxidized state, there are significant spectral differences between it and the native enzyme. These are believed (33) due to reduction of molybdenum from Mo(VI) to Mo(IV) and complexing of... 

The basis of this assay was first used to measure the activity of superoxide dismutase (SOD) using a xanthine/xanthine oxidase 02"-generating system. O2 generated via this enzyme will reduce feni (oxidised)-cytochrome c, but SOD (which has a much higher affinity for O2" than cytochrome c) will prevent this reduction. Babior, Kipnes and Cumutte (1973) modified this technique to provide a specific assay to measure O2 production by activated neutrophils. Thus, 02" reduces cytochrome c (measured by an absorbance increase at 550 nm), but this reduction will be blocked by the addition of exogenous SOD (Fig. 5.10). 

Xanthine oxidase (XO) was the first enzyme studied from the family of enzymes now known as the molybdenum hydroxylases (HiUe 1999). XO, which catalyzes the hydroxylation of xanthine to uric acid is abundant in cow s milk and contains several cofactors, including FAD, two Fe-S centers, and a molybdenum cofactor, all of which are required for activity (Massey and Harris 1997). Purified XO has been shown to use xanthine, hypoxan-thine, and several aldehydes as substrates in the reduction of methylene blue (Booth 1938), used as an electron acceptor. Early studies also noted that cyanide was inhibitory but could only inactivate XO during preincubation, not during the reaction with xanthine (Dixon 1927). The target of cyanide inactivation was identified to be a labile sulfur atom, termed the cyanolyzable sulfur (Wahl and Rajagopalan 1982), which is also required for enzyme activity. 

When O2 is the electron acceptor, the reduction can occur in either two-electron steps with FADH2 as reductant and H2O2 as the product or in a one-electron manner with 02 as the product. In the latter case, the reduced form of the flavin could be either FADH2 or FAD Recent studies on the reaction of O2 with reduced xanthine oxidase has shown that reoxidation of the six-electron reduced enzyme by O2 proceeds initially with two sequential two-electron steps to form two moles of H2O2 and the two-electron reduced form of the enzyme. Oxidation of the two-electron reduced form by O2 then proceeds via two-sequential one-electron steps to form two moles of O2 and oxidized enzyme. The differential rate of O2 release is suggestive of one mole arising from the one-electron... 

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