Xanthine oxidase molybdenum complex center

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... 

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]. 

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.

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 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). 

These techniques are applicable only to paramagnetic Mo(V) centers, but the EPR parameters are extremely sensitive to coordination changes at the molybdenum center 17, 64). The molybdenum and ligand hyperfine splittings can provide additional information about the coordination environment of the molybdenum(V) species and the chemical reactions at the molybdenum center. EPR spectra from xanthine oxidase were first reported in 1959 by Bray et al. (65), and Bray and co-workers have continued to develop the application of EPR spectroscopy to molybdenum enzymes 17, 64). In 1966 it was shown (66) that mixing [Mo04] with dithiols produced EPR signals with (g) and (A( Mo)) values similar to those of xanthine oxidase. Only recently, however, have the structures of such thiolate complexes been determined (see Section IV.B.2.b). 39) and P (67) ENDOR spec-... 

Molybdenum is present in many enzymes and is often EPR detectable (except for Mo-nitrogenase, where it exists in a complex MoFeySg homocitrate cluster). Shown in Figure 9 is the Mo " " (f) X-band spectrum of one form of xanthine oxidase, called the Very Rapid form. Xanthine oxidase is a homodimer containing one Mo center, two Fe-S, centers and one molecule of flavin adenine dinucleotide per subunit. This enzyme catalyzes the oxidation of xanthine at the Mo center with subsequent reduction of O2 at the flavin center. In this enzyme Mo + exhibits a slightly rhombic EPR spectrum (g = 2.0252, gj, = 1.9550, g = 1.9495 gave < 2). 

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