Xanthine oxidase couplings

R. Hille and R.F. Anderson, Coupled electron/proton transfer in complex flavoproteins — solvent kinetic isotope effect studies of electron transfer in xanthine oxidase and trimethylamine dehydrogenase. J. Biol. Chem. 276, 31193-31201 (2001). 

Although molybdenum and tungsten enzymes carry the name of a single substrate, they are often not as selective as this nomenclature suggests. Many of the enzymes process more than one substrate, both in vivo and in vitro. Several enzymes can function as both oxidases and reductases, for example, xanthine oxidases not only oxidize purines but can deoxygenate amine N-oxides [82]. There are also sets of enzymes that catalyze the same reaction but in opposite directions. These enzymes include aldehyde and formate oxidases/carboxylic acid reductase [31,75] and nitrate reductase/nitrite oxidase [83-87]. These complementary enzymes have considerable sequence homology, and the direction of the preferred catalytic reaction depends on the electrochemical reduction potentials of the redox partners that have evolved to couple the reactions to cellular redox systems and metabolic requirements. 

The cofactors of both xanthine and aldehyde oxidases belong to the LMoVI(S)(0) subfamily (see Section IV). However, inactive dioxo forms, LMovi(0)2, of both xanthine and aldehyde oxidase are known. These dioxo forms do not catalyze oxidation of the respective substrates of these enzymes. The Mov/Molv redox potential for the inactive bis(oxido) form of xanthine oxidase differs from the oxido-sulfido form by -30 mV (bovine xanthine oxidase) and -lOOmV (chicken liver xanthine oxidase) [91]. Although the difference is small, given the xanthine/uric acid reduction potential (-360 mV), it is possible that the Mov/MoIV couple (-433 mV) of the chicken-liver xanthine oxidase bis(ox-ido) form impedes the effective oxidation of xanthine for redox reasons alone. However, the bis(oxido) form of bovine xanthine oxidase (with a reduction potential of -386 mV) should be able to oxidize xanthine, since the redox potential, and hence the thermodynamic driving force, is sufficient for activity [91,92,99]. As substrate oxidation does not occur, the chemical differences between the bis(oxido) and oxido-sulfido (Movl) forms must be critical to the dramatic difference in activity (see Section VI.E.l). 

Shown in Figures 5-7 are the redox pathways for xanthine oxidase, sulfite oxidase, and nitrate reductase (assimilatory and respiratory), respectively. These schemes address the electron and proton (hydron) flows. The action of the molyb-doenzymes is conceptually similar to that of electrochemical cells in which half reactions occur at different electrodes. In the enzymes, the half reactions occur at different prosthetic groups and intraprotein (internal) electron transfer allows the reactions to be coupled (i.e., the circuit to be completed). In essence, this is the modus operandi of these enzymes, which must be determined before intimate mechanistic considerations are seriously addressed. 

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],... 

An additional crucial piece of information emerges from the alloxan-thine study (24). Thus, it was shown that one alloxanthine binds to the enzyme per active molybdenum site. This result clearly implies that the molybdenum site is mononuclear. If a dinuclear site were involved, then it would be unlikely to require two alloxanthine molecules for inhibition and would be expected to be at least partially inhibited with one alloxan-thine/two molybdenum. Also, a difference in binding constant would be expected for the second compared with the first bound alloxanthine, but none is found. This result, coupled with the lack of evidence for Mo(V)-Mo(V) spin-spin interactions in the EPR spectra, clearly implicates a mononuclear site, and it would seem that xanthine oxidase possesses two full catalytic units, each containing one molybdenum, one flavin, and two Fe2S2 units (20). Other molybdenum oxidases also contain paired prosthetic groups and subunits, and it is likely that they each have two catalytic units per molecule. 

The other molybdenum enzymes each contain duplicate prosthetic groups and paired subunits in addition to two molybdenum atoms. Many of the experiments performed for xanthine oxidase have also been carried out with aldehyde oxidase and sulfite oxidase, and there is no evidence for chemical Mo-Mo coupling in these enzymes. Thus, in oxidases, the evidence for mononuclear molybdenum sites appears strong, and in view of the duplicate subunits and composition found, it is reasonable to assume a similar situation in reductases as well. However, at present, insufficient information bars a full generalization. 

Figure 5. Proposed coupled proton-electron transfer scheme for xanthine oxidase activity (66, 67, 68)...

Many experimental observations on xanthine oxidase activity are correlated by this scheme, and at present, there appear to be no major inconsistencies. The coupled proton-electron transfer scheme (66, 67, 68) has been successfully incorporated into an overall mechanistic scheme (69) which explains, with great economy, a large amount of rather demanding data, from both kinetic and electron uptake experiments. 

The reaction was carried out in the following manner. Stock blood was transferred into test tubes, water was added, and the solution was frozen and thawed to lyse the cells. At zero time, excess xanthine oxidase was added as a coupling enzyme to convert all the hypoxanthine that was formed during the reaction to uric acid. The reaction was started by the addition of inosine in a phosphate buffer. 

Greenwood, R. J., Wilson, G. L., Pilbrow, J. R., and Wedd, A. G., 1993, Molybdenum(V) sites in xanthine oxidase and relevant analog complexes comparison of oxygen-17 hyperfine coupling, J. Am. Chem. Soc. 115 5385n5392. 

Lowe, D. J., and Bray, R. C., 1978, Magnetic coupling of the molybdenum and iron-sulfur centres in xanthine oxidase and xanthine dehydrogenase, Biochem. J. 169 4719 479. 

With the exception of the recently reported DMSO reductases from bacteria (71,72), all of the enzymes of Table I contain additional redox active prosthetic groups besides Mo-co. Substrate oxidation (reduction) occurs at the molybdenum center, and electrons are removed (added) via one of the other prosthetic groups. These two processes are coupled by intramolecular electron transfer between the molybdenum center and the other redox centers of the enzyme. Results for xanthine oxidase and sulfite oxidase and approaches to modeling the coupling in sulfite oxidase are summarized below. 

The salvage pathway does not involve the formation of new heterocyclic bases but permits variation according to demand of the state of the base (B), i.e. whether at the nucleoside (N), or nucleoside mono- (NMP), di- (NDP) or tri- (NTP) phosphate level. The major enzymes and routes available (Scheme 158) all operate with either ribose or 2-deoxyribose derivatives except for the phosphoribosyl transferases. Several enzymes involved in the biosynthesis of purine nucleotides or in interconversion reactions, e.g. adenosine deaminase, have been assayed using a method which is based on the formation of hydrogen peroxide with xanthine oxidase as a coupling enzyme (81CPB426). 

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