Xanthine oxidase molybdenum

It is usually believed that NO inhibits enzymes by reacting with heme or nonheme iron or copper or via the S-nitrosilation or oxidation of sulfhydryl groups, although precise mechanisms are not always evident. By the use of ESR spectroscopy, Ichimori et al. [76] has showed that NO reacts with the sulfur atom coordinated to the xanthine oxidase molybdenum center, converting xanthine oxidase into a desulfo-type enzyme. Similarly, Sommer et al. [79] proposed that nitric oxide and superoxide inhibited calcineurin, one of the major serine and threonine phosphatases, by oxidation of metal ions or thiols. 

AO/XO Aldehyde oxidase/xanthine oxidase (molybdenum hydroxylases)... 

The first indication of an essential metabolic role for molybdenum was obtained in 1953, when it was discovered that xanthine oxidase, important in purine metabolism, was a metalloenzyme containing molybdenum. Subsequently the element was shown to be a component of two other enzymes, aldehyde oxidase and sulphite oxidase. The biological functions of molybdenum, apart from its reactions with copper (see p. 123), are concerned with the formation and activities of these three enzymes. In addition to being a component of xanthine oxidase, molybdenum participates in the reaction of the enzyme with cytochrome C and also facilitates the reduction of cytochrome C by aldehyde oxidase. 

Molybdenum. Molybdenum is a component of the metaHoen2ymes xanthine oxidase, aldehyde oxidase, and sulfite oxidase in mammals (130). Two other molybdenum metaHoen2ymes present in nitrifying bacteria have been characteri2ed nitrogenase and nitrate reductase (131). The molybdenum in the oxidases, is involved in redox reactions. The heme iron in sulfite oxidase also is involved in electron transfer (132). 

Complexes of molybdenum and tungsten with bidentate sulfur ligands have been investigated extensively. In recent years, the work in this field has been escalated by the impetus of designing models of such bioinorganic enzymes as nitrogenase and xanthine oxidase (125). The early work reviewed by Coucouvanis (1) dealt exclusively with the isolation of oxomolybdenum(V) and -(VI) species. 

Romao MJ, Huber R (1998) Structure and Function of the Xanthine-Oxidase Family of Molybdenum Enzymes. 90 69-96 Rosenzweig A, see Penneman RA (1973) 13 1-52... 

The enzymes that utilize molybdenum can be grouped into two broad categories (1) the nitrogenases, where Mo is part of a multinu-clear metal center, or (2) the mononuclear molybdenum enzymes, such as xanthine oxidase (XO), dimethyl sulfoxide (DMSO) reductase, formate dehydrogenase (FDH), and sulfite oxidase (SO). The last... 

Another Mossbauer study on molybdenum hydroxylases was performed on a nonenriched sample of milk xanthine oxidase (219), and an unusually large AEq (3.2 mm/s at 175 K) was also observed for the ferrous site of one of the clusters. 

V. Milk Xanthine Oxidase Studies on the Role of Molybdenum. 117... 

Of the mammalian enzymes, the sulphite oxidase of bovine liver has only recently been discovered to contain molybdenum (15). The better known molybdenum enzymes, xanthine oxidase from cows milk (31) and aldehyde oxidase from rabbit liver (16) are closely related to one another as they are to the xanthine dehydrogenases from chicken liver (17) and from bacteria (18). 

Of experimental methods for studying the metal in enzymes, light absorption in the visible region from molybdenum chromophores is likely to be weak and frequently masked by stronger absorption from other enzyme constituents. Indeed only recently has a small molybdenum contribution to the absorption spectrum of even the most studied of these enzymes, xanthine oxidase, been demonstrated 33, see Section V F). 

Studies bearing on the role of molybdenum in enzymes will be exemplified by a detailed summary of results on the most studied of these enzymes, which is undoubtedly milk xanthine oxidase. To put this in its context, it will be preceded by a review of the general properties of xanthine oxidase. The final section will then be a short account of work on some of the other molybdenum enzymes. 

The feature of xanthine oxidase which is no doubt of the greatest chemical interest, is the presence of several non-protein components. Much effort has been expended in attempting to elucidate the respective roles of iron, flavin and molybdenum in the various enzyme catalysed reactions. Numerous studies of the iron constituent have been made of late (45, 46, 47, 48, 49, 50), it having been found to be of the iron-sulphur (51 a, 51 b) type. Neither iron (19) nor molybdenum (31) can be removed reversibly from the enzyme, though the FAD can be (52, see below). 

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 ... 

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...

Since studies bearing on the role of molybdenum in milk xanthine oxidase have depended heavily on the EPR method, it is convenient to precede detailed discussion by a general description of the various molyb-denum(V) EPR signals which have been obtained from the enzyme. 

Scheme 2. Species responsible for Mo(V) signals from xanthine oxidase. The diagram illustrates typical treatments which may be used to obtain the various molybdenum EPR signals from milk xanthine oxidase. Nomenclature of the signals is that of reference (78) and of the enzyme species, that of reference (19). Conditions for signal development refer to pH 8.2 and 20—25 0 with about 0.1 mM enzyme unless otherwise stated...

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.)...

As has been mentioned above, integrated signal intensities for molybdenum have always been less than 1 g. atom of Mo(V) per mole of xanthine oxidase. However, there are indications from recently performed integrations (90) that observed intensities of the Inhibited signal, in which Mo(V) is known to be stabilized (81), can be accounted for quantitatively when due allowance is made for the other species present in the samples. If this is confirmed it should make possible final rejection of earlier suggestions (87) that the enzyme contains two interacting molybdenum atoms in a single active centre. It should also help to eliminate possibilities (cf. 78) that only one of the two molybdenum atoms of the molecule is ever detected by EPR spectroscopy. 

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... 

Fig. 6. Difference spectra between xanthine oxidase inactivated with various pyra-zolo [3, 4-d] pyrimidines and the native enzyme. The spectra are believed to represent the increase in absorption occurring when Mo(VI) of native enzyme is converted to Mo(IV) complexed with the inhibitors. Spectra were obtained by treating the enzyme with inhibitors in the presence of xanthine, then admitting air, so as to re-oxidize the iron and flavin chromophores. The extinction coefficients, de, are expressed per mole of enzyme flavin. Since some inactivated enzyme was present, extinction coefficients per atom of molybdenum of active enzyme will be about 30% higher than these values. (Reproduced from Ref. 33, with the permission of Dr. V. Massey.)...

---