Xanthine oxidase Rapid signals

In the original rapid-freezing work on xanthine oxidase (53) it was found that in experiments employing about 1 mole of xanthine per mole of enzyme and an excess of oxygen, the time sequence of appearance of the various EPR signals was molybdenum (V), followed by flavin semi-quinone radical (FADH), followed by iron. This suggested that the electron transfer sequence might be ... 

Fig. 3. Rapid Mo(V) EPR signals obtained on reducing xanthine oxidase at pH 10 with 15 moles of xanthine for 1 min. at about 20 °. The upper four spectra are at 9.1 GHz and the lower four at 34.4 GHz. a, a, c, 8 refer to H2O as solvent and b, b, d, d to D2O. a, b, c, d are computer simulations of the experimental spectra, a, b, c, d, respectively. The interpretation is that two species, each having exchangeable protons which interact with Mo(V), are responsible for the signals. For one of these (dotted complex type II) there are two equivalent interacting protons and for the other (dashed complex type I), two non-equivalent protons. These species are believed to correspond to two different complexes of reduced xanthine oxidase with xanthine. (Reproduced from ref. 78 see also Table 2 for the parameters of the signals.)...

Fig. 4. Anaerobic titration of xanthine oxidase with xanthine at pH 8.2 with a reaction time of 2 min. at about 20°. The integrated intensity of the Rapid molybdenum EPR signals (in arbitrary units) is plotted against the number of moles of xanthine added per mole of active enzyme. Activity/A4jo for the enzyme samples used was 112 corresponding to an active enzyme content of 57%. Thus the molar ratios of xanthine/total xanthine oxidase have been multiplied by 1.76 to refer to the active form only. Some of the EPR spectra (recorded at about — 130° and 9.3 GHz) are reproduced to show the changes in signal type as the amount of xanthine is increased. (Data re-calculated from ref. 88, with intensities corrected for variations in tube diameter and enzyme concentration calculated in terms of active enzyme.)...

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.)...

An EPR signal, characteristic for the superoxide radical, was observed by the rapid-freezing technique in the oxidation at pH 10 of xanthine by dioxygen catalysed by xanthine oxidase (EC 1,2.3.2) The enzymatic reduction of dioxygen by aldehyde oxidase (EC 1.2.3.1) produces also the superoxide radical. 

The decrease in rate of reaction of xanthine oxidase with the size of purine substrate is also consistent with a size-selective active site pocket and possible metal binding of substrate [242,243], A strongly coupled nitrogen is not observed in the very rapid signal, which is thought to include bound product, so it would appear that the urate is not N bound to the molybdenum center [152-158],... 

Fig. 26 (149). EPR signals of O - obtained both enzymically and non-enzymically. Recording at —170 °C, rapid freezing technique (152) a) xanthine—xanthine oxidase reaction. Note the molybdenum signals (overmodulated). The sloping base line is attributed to an iron signal b) non-enzymically induced Og- from H2O2 and NaIC>4, pH 9.9 c) same as (b) but at pH 13.2...

Figure 3.8 Orientation dependent Q-band CW ENDOR spectra of C-labeled sample of HMP-gene rated very rapid signal of xanthine oxidase across the EPR envelope. (A) Experimental spectra. Conditions v w = 35.165 GHz, temperature 2 K. (B) Corresponding simulated ENDOR spectra. Only the largest nuclear frequency v+ is given for clarity. The parameters used for the simulation are given in Table 3.4 Reprinted with permission from ref. 43. Copyright (2001) American Chemical Society.

Canne and co-workers have presented EPR studies of three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline l-oxidoreductaseJ In quinoline 2-oxidoreductase a neutral flavin radical was observed, while in quinaldine 4-oxidase an anionic radical was detected. The rapid Mo(V) signal was observed in all three enzymes with only small differences in magnetic parameters. From spectra simulations of Mo (/ = 5/2) substituted quinoline 2-oxidoreductase, a deviation of 25° between the maximal g and Mo-hfc tensor component was derived. The Mo(V) species was detected in small amounts upon reduction with substrates in quinoline 2-oxidoreductase and quinaldine 4-oxidase, but showed a different kinetic behaviour with an intense EPR signal in isoquinoline 1-oxidoreductase. The two [2Fe-2S] clusters produced different EPR signals in all three enzymes and, in isoquinoline 1-oxidoreductase, revealed a dipolar interaction, from which a maximum distance of 15 A was estimated. 

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