Introduction Xanthine oxidoreductase (XOR) is a molybdoflavin enzyme that catalyzes the terminal two reactions in purine degradation in primates; oxidation of hypoxanthine to xanthine and the subsequent oxidation of xanthine to uric acid. XOR is a homodimer of ~300 kD with each subunit consisting of four redox centers: a molybdenum cofactor (Mo-co), one FAD site and two Fe/S clusters, Fig. 1. The Mo-co is the site of purine oxidation while NAD+ and O2 reduction occur at the FAD. The two Fe/S clusters provide the conduit for electron flux between the Mo-co and the FAD [1–3]. The enzyme is transcribed as a single gene product, xanthine dehydrogenase (XDH) where substrate-derived electrons reduce NAD+ to NADH, Fig. 1A. However, during inflammatory conditions, oxidation of key cysteine residues (535 and 992) and/or limited proteolysis converts XDH to xanthine oxidase (XO) . In the oxidase form, affinity for NAD+ at the FAD is greatly decreased while affinity for oxygen is significantly enhanced resulting in univalent and divalent electron transfer to O2 generating O2 •− and hydrogen peroxide (H2O2), respectively, Fig. 1B . This capacity to reduce O2 led to XOR being identified as the first source of biological O2 •− formation and subsequently as a significant source of reactive species mediating ischemia/reperfusion injury [6,7]. As the redox field progressed, several additional enzymatic and non-enzymatic sources of free radicals and reactive species have been identified yet, to date, XOR remains the most pharmacologically targetable thus incentivizing extensive exploration of inhibition strategies to address disease processes where elevated rates of reactive species formation are contributory. Oxidant formation Most reports refer to XO as a source of O2 •− and assume H2O2 formation is a result of spontaneous dismutation of O2 •−. This premise is completely invalid as attainment of 100% O2 •− generation requires XO turnover at pH 10.0 in an environment of 100% O2 . However, under room air and pH 7.4, XO transfers over 72% of its substrate-derived electrons to O2 divalently to generate H2O2 and thus 28% to O2 •− formation. This observation is critically important as it clearly demonstrates that, under conditions approaching those encountered in vivo, H2O2 is the major reactive product of XO-catalyzed O2 reduction [8,9]. The prime determinate of the relative quantities of O2 •− and H2O2 generated by XO is O2 tension. For example, at pH 7.4 and 10% O2 XO generates ~26% O2 •− and thus ~74% H2O2 whereas at 1% O2, XO forms ~90% H2O2 and only ~10% O2 •−, Fig. 2 . In addition to O2 tension, pH and purine concentration also play a significant role in divalent versus univalent electron transfer to O2. The reaction of hypoxanthine/xanthine at the Mo-co of XO is based-catalyzed with a pH optimum of 8.9 and a K m =~6.5 µM. Under normal physiologic conditions, hypoxanthine + xanthine levels in humans are ~1–3 µM; however, under hypoxic/inflammatory conditions these levels have been reported as high as 50–100 µM while pH concomitantly drops below 7.0 [10–12]. When this occurs, total purine (hypoxanthine+xanthine) concentration is well above the K m and thus will not significantly impact either rates of electron deposition at the Mo-co or resultant transfer to the FAD. However, acidic pH will significantly retard purine–Mo-co reaction thereby reducing the electron flux rate which favors divalent transfer to O2 to generate H2O2. Therefore, under ischemic and/or hypoxic conditions, where both O2 levels and pH are reduced, H2O2 formation is favored suggesting that XO activity may be influential in numerous signaling cascades where H2O2 has been noted to participate. However, this hypoxia-mediated proclivity for H2O2 production cannot overshadow the fact that rates of O2 •− formation by XO under these same conditions are sufficient to mediate alterations in vascular function by reducing •NO bioavailability via direct reaction (•NO + O2 •−→ONOO−) [13–15]. While the post-translational conversion of XDH to XO has become synonymous with conversion from a source of reducing equivalents to a source of reactive oxygen species (ROS), it is important to recognize that under certain circumstances XDH effectively reduces O2 to generate ROS. Although NAD+ is the preferred electron acceptor for XDH, when levels of this substrate are low XDH will utilize O2 . These conditions include hypoxia either localized, regional or systemic where O2-dependent alterations in cellular respiration lead to decreased mitochondrial NADH oxidation and thus significant diminution of NAD+ levels . This being said, care should be taken not to exclusively associate XDH with the form of XOR that does not produce ROS. XO-endothelial interaction In humans, XOR is ubiquitously expressed with the liver and intestines displaying the highest specific activity . Hypoxia as well as inflammatory cytokines (TNF-α, IL-1β, IFN-γ), induce XDH expression in tissues and vascular endothelial cells where it is released to the circulation, Fig. 2 [18,19]. Circulating XDH is rapidly (<1 min) converted to XO where it avidly binds to negatively charged glycosaminoglycans (GAGs) on the apical surface of vascular endothelial cells [20,21]. This XO–endothelium interaction is exemplified in animal models and clinical studies of cardiovascular disease where intravenous administration of heparin results in a substantive increase in plasma XO activity, suggesting heparin-mediated mobilization of XO from vascular endothelial GAGs [21–23]. While XO exhibits a net negative charge at physiological pH, pockets of cationic amino acid motifs on the surface of the protein result in high affinity for GAGs (K d =6 nM) [21,24,25]. Binding to and sequestration of XO on GAGs: (1) amplifies local XO concentration and subsequent ROS generation; (2) alters XO kinetic properties further shifting oxidant formation from O2 •− to H2O2 and (3) confers significant resistance to inhibition from the pyrazalopyrimidine-based inhibitors, allo/oxypurinol . For example, when compared to XO in free in solution, XO–GAG association decreases substrate binding affinity and thus: (1) increases the K m for xanthine over 3-fold (6.5→21.2 µM); (2) reduces O2 •− production by 34% favoring H2O2 formation and (3) induces a 5-fold increase in the K i for allo/oxypurinol (85→451 nM) . Taken together, inflammation-mediated up-regulation of XDH, export to the circulation, rapid conversion to XO and sequestration by the endothelium coalesce to generate a vascular milieu favoring increased rates of reactive species generation that can participate in mediating the loss of homeostasis, Fig. 2. This deleterious action of XO has been noted in various reports of vascular and cardiopulmonary diseases including heart failure, chronic obstructive pulmonary disease (COPD), pulmonary hypertension, sickle cell disease and Type I and II diabetes [14,27–30]. XOR knockouts and inhibition strategies For an enzyme whose activity was described in 1889 followed by it being named xanthine oxidase in 1901 and first purified in 1939, surprisingly little detail is known regarding its regulation and subsequent interplay with biomolecular pathways when compared to other enzymes with much more recent history . Potential reasons for this discrepancy in understanding include: (1) lethality of global XDH knockouts; (2) absence of reports utilizing tissue-specific conditional knockouts; (3) side-effects resulting from pharmacological knockdown with tungsten supplementation; (4) promiscuity of the active site resulting in ambiguity regarding both substrate identity and inhibition by compounds designed to specifically target other molybdopterin enzymes and (5) resistance to inhibition by allo/oxypurinol conferred by binding to vascular endothelial GAGs. Attempts to establish homozygous knockouts of XDH in mice have resulted in the death of pups before 30 days of age due to kidney fibrosis and failure attributed to excessive hypoxanthine deposition [32,33]. Similar effects were obtained with heterozygous XDH knockouts where both nutrient absorption and kidney failure resulted in death in a similar timeframe as XDH−/−. These unfortunate side-effects have relegated investigators to utilize allo/oxypurinol-based inhibition or global XOR knockdown with dietary tungsten (W) supplementation for proof-of-principle experimentation. Dietary supplementation with sodium tungstate (NaW) results in replacement of the active site Mo with W producing an enzyme that is inactive with respect to hypo/xanthine oxidation to uric acid. However, it is important to note that W-mediated inactivation of the Mo-co does not affect the capacity of the FAD in XOR to be reduced by NADH and subsequently react with and reduce O2 to produce O2 •− and H2O2. In addition, treatment with NaW also inactivates other members of the molybdopterin family including aldehyde oxidase, sulfite oxidase and mitochondrial amidoxime reducing component 1 (MARC1) which can lead to significant ambiguity regarding interpretation of results. On the other hand, inhibition of XOR with allo/oxypurinol is also not optimal as: (1) allopurinol can mediate effects on other purine catabolic pathways including those resulting in alteration of adenosine levels ; (2) reaction of allopurinol with XO induces enzyme turnover resulting in O2 •− and H2O2 formation  and (3) plasma allo/oxypurinol concentrations (100–400 µM) well above those tolerated clinically (30–90 µM) are incapable of fully inhibiting XO when it is sequestered by vascular GAGs [26,35]. As a result of these limitations we have recently identified febuxostat (Uloric) to be more optimal for exploring contributions of XOR both in vivo and tissue culture. For example, febuxostat concentrations (25–50 nM) well below the reported plasma C max (15 µM) for the clinic demonstrate over 3 orders of magnitude greater potency than allopurinol (K i =0.9 nM vs. 1.6 µM), are not affected by XOR-vascular GAG association and do not alter other purine catabolism pathways [34,35]. In toto, these findings clearly demonstrate the potential benefit of using febuxostat to interrogate XOR-dependence in various experimental models. XOR-catalyzed •NO production For decades, the dogma in the field has been as described above; specifically that inflammation/hypoxia-induced enhancement of XO activity equates to elevated rates of XO-derived ROS generation, propagation/exacerbation of the disease process and ultimately poor clinical outcomes. This correlation has been substantiated in several disease models where XO inhibition leads to a reduction in symptoms and measurable restoration of function. However, recent reports have posed a bold challenge to the standing paradigm by demonstrating a nitrate/nitrite (NO2 −) reductase function for XOR (1e− reduction of NO2 − to •NO) suggesting XOR to be a source of beneficial •NO under these same hypoxic/inflammatory conditions. In essence, these observations directly countervail a substantive body of literature indicating XO inhibition to be beneficial and as such affirm the need to more closely interrogate XOR-catalyzed reactions and potential factors that alter product formation. For example, reduction of NO2 − to •NO is indeed catalyzed by purified XO under anoxic conditions when electrons are supplied by either xanthine or NADH [36–38]. Nitrite reduction occurs at the reduced Mo-co (Mo-co IV) and electrons driving this reaction can be supplied directly by xanthine (Fig. 1C) or indirectly by NADH via electron donation at the FAD with subsequent retrograde flow to the Mo-co, Fig. 1D . At this point, it is critical to note that work with the purified enzyme has revealed two issues requiring resolution before the biological relevance of XOR-derived •NO can be substantiated. First, the NO2 − reductase activity of XOR is inhibited by O2 which results from oxidation of the Mo-co mediated by electron withdrawal from the FAD . Second, the affinity for NO2 − at the Mo-co of XOR is 3 orders of magnitude less than for xanthine (K m -NO2 −=2.5 mM vs. K m -xanthine=6.5 µM) . Despite these formidable issues, several reports demonstrate significant reduction in or ablation of salutary outcomes attributable to NO2 − treatment upon inhibition of XOR activity with allo/oxypurinol affirming the need for more vigorous investigation to fully elucidate this reductive process. For example, systemic inhibition of XOR activity has diminished protective effects of NO2 − treatment in models of intimal hyperplasia , lung injury [42,43], myocardial infarction , pulmonary hypertension  and ischemia/reperfusion (I/R)-induced damage [46–49]. It is also important to note that plasma levels of NO2 − are reported to be enhanced in an XOR-dependent manner by treatment with nitrate (NO3 −) where XOR serves first as a NO3 − reductase (NO3 −+1e−→NO2 −) and ultimately a NO2 − reductase (NO2 −+1e−→•NO). This XO-catalyzed process was described over 50 years ago  and recently expanded to in vivo models . In these experiments treatment of germ-free mice (void of bacterial NO3 − reductases) with NO3 − resulted in elevation of plasma NO2 − levels that were not observed when mice received co-treatment with allopurinol and thus are consistent with previous biochemical reports demonstrating NO3 − reductase activity for XOR . In the aggregate, there is a new body of evidence suggesting a protective role for XOR under hypoxic and inflammatory conditions in the presence of elevated levels of NO2 −, summarized in Fig. 3. However, several key issues remain unclear regarding the microenvironmental conditions necessary for operative and biologically relevant nitrite reductase activity of XOR in vivo and were recently extensively reviewed . Although XOR has been studied for 114 years, it is clear from the information provided herein that we have only begun to understand the complexity regarding the interplay between crucial microenvironmental factors and the identity/generation of XOR-derived reactive products as well as their impact on cellular signaling both in normal and pathophysiology. Suffice it to say the long-standing dogma identifying XDH as a housekeeping enzyme and XO as a mediator of negative clinical outcomes is beginning to crumble as we uncover new roles for XOR in the network of adaptive responses that serve to maintain homeostasis.