Xanthine oxidase molybdenum center

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. 

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... 

XOD is one of the most complex flavoproteins and is composed of two identical and catalytically independent subunits each subunit contains one molybdenium center, two iron sulfur centers, and flavine adenine dinucleotide. The enzyme activity is due to a complicated interaction of FAD, molybdenium, iron, and labile sulfur moieties at or near the active site [260], It can be used to detect xanthine and hypoxanthine by immobilizing xanthine oxidase on a glassy carbon paste electrode [261], The elements are based on the chronoamperometric monitoring of the current that occurs due to the oxidation of the hydrogen peroxide which liberates during the enzymatic reaction. The biosensor showed linear dependence in the concentration range between 5.0 X 10 7 and 4.0 X 10-5M for xanthine and 2.0 X 10 5 and 8.0 X 10 5M for hypoxanthine, respectively. The detection limit values were estimated as 1.0 X 10 7 M for xanthine and 5.3 X 10-6M for hypoxanthine, respectively. Li used DNA to embed xanthine oxidase and obtained the electrochemical response of FAD and molybdenum center of xanthine oxidase [262], Moreover, the enzyme keeps its native catalytic activity to hypoxanthine in the DNA film. So the biosensor for hypoxanthine can be based on... 

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. 

A large number of studies devoted to metal-sulfur centers are motivated by the occurrence of such arrangements at the active site of various metalloenzymes [1-13]. Mononuclear complexes with Mo=0 func-tion(s) and possessing sulfur ligands in their coordination sphere have been extensively investigated since they can be seen as models of the active site of enzymes such as nitrate- and DM SO reductases or sulfite- and xanthine oxidases [1-4]. On the other hand, a large variety of mono-, di-, and polynuclear Mo—S centers have been synthesized in order to produce functional models of the Mo-nitrogenase since the exact nature (mono-, di- or polynuclear) of the metal center, where N2 interacts within the iron-molybdenum cofactor (FeMo—co) of the enzyme is still unknown [4-8]. 

The rationale for studies on flavin semiquinone metal interactions stems from the presence of flavin coenzymes which participate in electron transfer in a number of metalloflavoproteins. Iron-containing redox centers such as the heme and nonheme iron sulfur prosthetic groups (Fe2/S2, Fe+ZS, or the rubredoxin-type of iron center) constitute the more common type of metal donor-acceptor found in metalloflavoproteins, although molybdenum is encountered in the molybdenum hydroxylases (e.g. xanthine oxidase, aldehyde dehydrogenase). 

Barber, M. J., Siegel, L. M. Proton and electron affinities and magnetic interactions associated with the molybdenum flavin, and iron-sulfur centers of milk xanthine oxidase. In Flavins and flavoproteins (Massey, V., Williams, C. H. eds.) pp. 796-804, New York, Amsterdam, Oxford, Elsevier/North Holland 1982... 

Purine nucleotides are degraded by a pathway in which they lose their phosphate through the action of 5 -nucleotidase (Fig. 22-45). Adenylate yields adenosine, which is deaminated to inosine by adenosine deaminase, and inosine is hydrolyzed to hypoxanthine (its purine base) and D-ribose. Hypoxanthine is oxidized successively to xanthine and then uric acid by xanthine oxidase, a flavoenzyme with an atom of molybdenum and four iron-sulfur centers in its prosthetic group. Molecular oxygen is the electron acceptor in this complex reaction. 

Figure 16-31 (A) Structure of molybdopterin cytosine dinucleotide complexed with an atom of molybdenum. (B) Stereoscopic ribbon drawing of the structure of one subunit of the xanthine oxidase-related aldehyde oxidoreductase from Desulfo-vibrio gigas. Each 907-residue subunit of the homodimeric protein contains two Fe2S2 clusters visible at the top and the molybdenum-molybdopterin coenzyme buried in the center. (C) Alpha-carbon plot of portions of the protein surrounding the molybdenum-molybdopterin cytosine dinucleotide and (at the top) the two plant-ferredoxin-like Fe2S2 clusters. Each of these is held by a separate structural domain of the protein. Two additional domains bind the molybdopterin coenzyme and there is also an intermediate connecting domain. In xanthine oxidase the latter presumably has the FAD binding site which is lacking in the D. gigas enzyme. From Romao et al.633 Courtesy of R. Huber.

Protein sequence homology suggests that sulfite oxidase and assimilatory nitrate reductase are members of the same molybdenum enzyme subfamily [31]. Consistent with this classification, the cofactors of sulfite oxidase and assimilatory nitrate reductase differ significantly from those in dmso reductase, aldehyde oxido-reductase, xanthine oxidase (see Section IV.E.), and even respiratory nitrate reductase (Section IV.D). The EXAFS of both sulfite oxidase [132-136] and assimilatory nitrate reductase [131,137,138] and x-ray studies of sulfite oxidase (chicken liver) [116] confirm that the molybdenum center is coordinated by two sulfur atoms from a single MPT ligand and by the sulfur atom of a cysteine side chain. The Movl state is bis(oxido) coordinated (Figure 14). 

It has long been a synthetic challenge to prepare mono(oxido) mono(sulfido) molybdenum centers that are analogous to the Mo(VI) state of enzymes in the xanthine oxidase family. Although studies in this area have met with limited success, it seems possible that the sulfido linkage of these centers could be stabilized by the 1,2-enedithiolate or by a molybdenum-bound thiolate ligand. 

Mechanisms of action for the metal centers in acetylene hydratase, polysulfide reductase, and formate dehydrogenase have been briefly described in Sections VI.A and VLB. The discussion, in each case, was relatively straightforward insofar as the natures of these reactions lend themselves to simple mechanistic proposals. The mechanism by which the metal centers function in most of the other Mo and W enzymes is not as obvious. We elect to discuss mechanistic roles for the molybdenum centers in xanthine oxidase, sulfite oxidase, and dmso reductase. These enzymes are representative members of each large class of molybdenum enzymes, and the large body of literature on each enzyme makes detailed discussion possible. 

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],... 

There is, however, more direct evidence for the presence of Mo (IV) in the cycle of xanthine oxidase. This evidence comes from the experiments of Massey and co-workers (24) who used alloxanthine (l) to trap the enzyme in its reduced state. A strong complex is formed between the reduced enzyme and alloxanthine, and excess alloxanthine and reductant can be removed. The enzyme is then reoxidized with Fe(CN)63", and two electrons per molybdenum center are found after the electrons required for the reoxidation of the iron-sulfur and flavin groupings are... 

It is obvious that x-ray cyrstallographic methods will be the final arbiter of the structural features of molybdoproteins, but until such structures are obtained, and even afterwards as far as dynamic features are concerned, spectroscopic methods must be used to gain insight into the nature of these catalysts. Electronic spectroscopy so far has been of little use here since molybdenum complexes in general appear to exhibit broad weak absorptions. In proteins these are always buried under absorptions from hemes, flavins, and iron-sulfur centers. Massey et al., (15) discovered that pyrazolo [3,4-d] pyrimidines will bind Mo (IV) in milk xanthine oxidase that had been reduced with xanthine... 

Xanthine Oxidase. This molybdoenzyme is readily available from cows milk in gram quantities (28) and is relatively stable, which accounts for the fact that it is by far the most intensively studied molybdoenzyme. Bray and Swann (5) have reviewed comprehensively the earlier literature, and recent papers by Olson et al. (20) summarize combined kinetic and thermodynamic approaches to the states of the prosthetic groups during catalysis. Two molybdenum, four iron-sulfur centers, and two FAD groups are present in each molecule. An important point raised by Edmondson, et al. (29) is that the rates of internal electron transfer among the prosthetic groups appear to be much more rapid than turnover. Olson et al., (20) deduced that the reduction potentials of the two processes Mo(VI) <— Mo(V) <— Mo(IV) were —60 and —31 mv, respectively, relative to the redox potential for one of the iron-sulfur centers (center II) in the molecule. Thus, at equilibrium one can never have more than a small fraction of molybdenum as... 

Xanthine oxidase catalyzes the oxidation of hypox-anthine and xanthine to uric acid. Xanthine oxidase is a complex metalloflavoprotein containing one molybdenum, one FAD and two iron-sulfur centers of the ferredoxine type in each of its two independent subunits. Usually, the enzyme is isolated from cow s milk. The enzyme is inhibited by allopurinol and related compounds. The production of uric acid from the substrate (xanthine) can be determined by measuring the change in optical density in the UV range. 

As yet, no X-ray crystal structures are available for any of the molybdenum enzymes in Table I. Therefore, present descriptions of the coordination environment of the molybdenum centers of the enzymes rest primarily upon comparisons of the spectra of the enzymes with the spectra of well-characterized molybdenum complexes. The two most powerful techniques for directly probing the molybdenum centers of enzymes are electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS), especially the extended X-ray absorption fine structure (EXAFS) from experiments at the Mo K-absorption edge. Brief summaries of techniques are presented in this section, followed by specific results for sulfite oxidase (Section III.B), xanthine oxidase (Section III.C), and model compounds (Section IV). 

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