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Review
. 2013 Jun 10;1(1):353-8.
doi: 10.1016/j.redox.2013.05.002.

Xanthine oxidoreductase-catalyzed reactive species generation: A process in critical need of reevaluation

Nadiezhda Cantu-Medellin  1 , Eric E Kelley
Affiliations
Review

Xanthine oxidoreductase-catalyzed reactive species generation: A process in critical need of reevaluation

Nadiezhda Cantu-Medellin et al. Redox Biol. .

Abstract

Nearly 30 years have passed since the discovery of xanthine oxidoreductase (XOR) as a critical source of reactive species in ischemia/reperfusion injury. Since then, numerous inflammatory disease processes have been associated with elevated XOR activity and allied reactive species formation solidifying the ideology that enhancement of XOR activity equates to negative clinical outcomes. However, recent evidence may shatter this paradigm by describing a nitrate/nitrite reductase capacity for XOR whereby XOR may be considered a crucial source of beneficial (•)NO under ischemic/hypoxic/acidic conditions; settings similar to those that limit the functional capacity of nitric oxide synthase. Herein, we review XOR-catalyzed reactive species generation and identify key microenvironmental factors whose interplay impacts the identity of the reactive species (oxidants vs. (•)NO) produced. In doing so, we redefine existing dogma and shed new light on an enzyme that has weathered the evolutionary process not as gadfly but a crucial component in the maintenance of homeostasis.

Keywords: Free radicals; GAGs, glycosaminoglycans; H2O2, hydrogen peroxide; Hypoxia; I/R, ischemia/reperfusion; Inflammation; NOS, nitric oxide synthase; Nitric oxide; Nitrite; O2•−, superoxide; Oxygen tension; ROS, reactive oxygen species; XDH, xanthine dehydrogenase; XO, xanthine oxidase; XOR, xanthine oxidoreductase); Xanthine oxidoreductase; •NO, nitric oxide.

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Figures

Fig. 1
Fig. 1
XOR-Catalyzed Reactions. (A) For XDH, xanthine is oxidized to uric acid and electrons transferred via 2 Fe/S centers to the FAD where NAD+ is reduced to NADH. (B) For XO, xanthine is oxidized to uric acid and electrons are transferred to the FAD where O2 is reduced to O2•− and H2O2. Under normal O2 tension and pH the Mo-co would reside more often in the oxidized +6 (VI) valence as electrons are rapidly transferred to O2 at the FAD. (C) Nitrite (NO2−) undergoes a 1 electron reduction to •NO at the Mo-cofactor of XO (electrons are donated directly to Mo by xanthine). (D) NO2− is reduced to •NO at the of XO (electrons are supplied by NADH and transferred retrograde reducing the Mo). Under low O2 tensions and pH the Mo-co would reside more often in the reduced +4 (IV) valence as electrons are more slowly transferred to O2. This decrease in electron flux from the Mo-co to the FAD is depicted in (C) as diminished arrows whereas in (D) NADH-mediated electron donation at the FAD is out-competing O2-mediated electron withdrawal and thus the arrows are reversed indicating flux from the FAD to the Mo-co.
Fig. 2
Fig. 2
Hypoxic/inflammatory induction of XOR and vascular consequences. (Top) Inflammatory cytokines and/or hypoxia induce XDH transcription and resultant protein expression. In vascular endothelial cells XDH is exported to the circulation where it is rapidly converted to XO by plasma proteases. However, cellular export is not requisite for XDH conversion to XO as enhanced oxidative stress within the endothelium can induce oxidation of critical cysteine residues that mediate reversible conversion to XO. Once in the circulation, negatively charged glycosaminoglycans (GAGs) on the luminal surface of the endothelium bind and sequester XO by high affinity (Kd=6 nM) interaction with pockets of cationic amino acids on the surface of the enzyme. This sequestration amplifies local XO levels creating a vascular milieu whereby, in the presence of hypoxanthine and/or xanthine, enhanced rates of O2•− and H2O2 formation ensue. (Bottom) A key determinate regulating the relative amounts of O2•− and H2O2 generated by XO is the concentration of molecular O2. Shown is a cartoon representing the change in relative percentages of O2•− and H2O2 formed by XO at 10% O2 (~130 µM O2) compared to 1% O2 (~13 µM O2). This range of O2 tension is critically important as it represents from well above to 50% below the Km-O2 at the FAD-cofactor of 27 µM or ~2% O2. As the O2 tension drops below this Km value the FAD-cofactor assumes more time in the fully reduced FADH2 state where, upon reaction with O2, divalent electron transfer is preferred. This process assumes constant electron from the Mo-co (e.g. [hypoxanthine+xanthine] above the 6.5 µM Km at the Mo-co) which would be expected under conditions similar to those encountered in the lumen of an ischemic/hypoxic vessel. In addition, it is critical to note that XO–GAG association as well as acidic pH serves to further favor H2O2 formation. Taken together, moderate to severe hypoxia induces XDH expression, export and conversion to XO that is subsequently captured by GAGs in an environment the primed for catalyzing the formation of H2O2 as well as a little O2•−.
Fig. 3
Fig. 3
Hypoxic conversion of XOR from oxidant to •NO production. Hypoxia mediates the alteration of microenvironmental factors that coalesce to both facilitate the conversion of XO from oxidant to •NO production and diminish the capacity of eNOS to catalyze •NO formation as well as enhance its potential to uncouple and produce O2•−. These factors include: (1) acidic pH; (2) elevation of NADH levels; (3) oxidation of biopterin and, of course; (4) diminution of O2 tension. Nitrite reduction at the Mo-co of XO is acid catalyzed with a pH optimum ~6–6.5 while xanthine oxidation the Mo-co is base catalyzed with a pH optimum=8.9. Therefore, lower pH confers a reduction in affinity for xanthine while increasing affinity for NO2−. Thus, lower pH results in an environmental setting more favorable for NO2− to compete with xanthine for the Mo-co. This shift in affinity away from xanthine and toward NO2− is further augmented by reduction in O2 tension to values below the Km-O2 at the FAD (27 µM or ~2% O2). Once this occurs, electron withdrawal from the FAD slows resulting in the Mo-co assuming a more reduced state (Mo-co IV, see Fig. 1C and D) which is crucial for two reasons: (1) NO2− reduction requires a reduced Mo-co and (2) xanthine oxidation requires an oxidized Mo-co. Therefore, O2 tensions at or below 2% further assist the ability of NO2− to compete with xanthine for reaction at the Mo-co. As seen in Fig. 1D, hypoxia-mediated elevation of NADH levels can also further augment the potential for NO2− reduction at the Mo-co by competing with O2 for reaction at the FAD. In this case, NADH-FAD reaction results in reduction of the FAD to FADH2 inducing Mo-co reduction by retrograde electron flux as well as inhibition of O2-mediated electron withdrawal. On the other hand, this same inflammatory setting negatively impacts •NO formation by eNOS. For example, as O2 tensions fall below 2% (27 µM): (1) O2 becomes limiting as a substrate for eNOS-catalyzed •NO production where the Km-O2 for eNOS=23 µM and (2) acid pH coupled with elevated levels of oxidants drive eNOS uncoupling and the propensity for eNOS-mediated O2•− generation (depicted above the cell on the right in small font). Taken together, diminution of O2 tension, acidic pH, elevation of NADH levels, and oxidation of biopterin converge to generate an environment whereby the burden for •NO production shifts from eNOS to XOR. Furthermore, the critical O2 concentration where this shift or “switch” is triggered is assumed to be near 2% where the Km-O2 values for both XO and eNOS collide (depicted by a pivot point in the cartoon). However, it is crucial to note that if this process is to be of biological relevance then: (1) NO2− and/or NO3− levels must be significantly elevated by dietary or pharmacologic supplementation and (2) the proposed interplay between the components of these concerted reactions must be vigorously pursued and validated.
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