Health, Safety & Environment

Xanthine dehydrogenase and xanthine oxidase

Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy

Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo(VI) ion with each one MPT, Mo horizontal lineO, Mo-O-, and Mo horizontal lineS ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK approximately 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK approximately 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo-OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.

Reschke, S., Mebs, S., Sigfridsson-Clauss, K. G., Kositzki, R., Leimkuhler, S., and Haumann, M.,Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy, Inorg Chem, 2017, 56, 2165-2176.


Synthesis and characterization of a novel organic nitrate NDHP: role of xanthine oxidoreductase-mediated nitric oxide formation

In this report, we describe the synthesis and characterization of 1,3-bis(hexyloxy)propan-2-yl nitrate (NDHP), a novel organic mono nitrate. Using purified xanthine oxidoreductase (XOR), chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy, we found that XOR catalyzes nitric oxide (NO) generation from NDHP under anaerobic conditions, and that thiols are not involved or required in this process. Further mechanistic studies revealed that NDHP could be reduced to NO at both the FAD and the molybdenum sites of XOR, but that the FAD site required an unoccupied molybdenum site. Conversely, the molybdenum site was able to reduce NDHP independently of an active FAD site. Moreover, using isolated vessels in a myograph, we demonstrate that NDHP dilates pre-constricted mesenteric arteries from rats and mice. These effects were diminished when XOR was blocked using the selective inhibitor febuxostat. Finally, we demonstrate that NDHP, in contrast to glyceryl trinitrate (GTN), is not subject to development of tolerance in isolated mesenteric arteries.

Zhuge, Z., Paulo, L. L., Jahandideh, A., Brandao, M. C. R., Athayde, P. F., Lundberg, J. O., Braga, V. A., Carlstrom, M., and Montenegro, M. F.,Synthesis and characterization of a novel organic nitrate NDHP: Role of xanthine oxidoreductase-mediated nitric oxide formation, Redox biology, 2017, 13, 163-169.

Exploring the interaction between Salvia miltiorrhiza and xanthine oxidase: insights from computational analysis and experimental studies combined with enzyme channel blocking

Xanthine oxidase (XO) has emerged as an important target not only for gout but also for cardiovascular diseases and metabolic disorders involving hyperuricemia. Salvia miltiorrhiza, a traditional Chinese medicine, is widely used for the clinical treatment of cardiovascular diseases. Herein, an integrated approach consisting of computational analysis and experimental studies was employed to analyze the interaction between S. miltiorrhiza and XO. Molecular docking simulations were performed to reveal the binding characteristics of the chemicals identified in S. miltiorrhiza on the basis of the total docking score and key molecular determinants for binding. The affinities of 10 representative compounds from the herb to XO were predicted and then confirmed by enzyme inhibitory assay in vitro. The binding specificity of these compounds was further validated by enzyme channel blocking, which contributed to elucidating the interaction mechanisms between the herb and XO. Results suggested that several compounds in S. miltiorrhiza exhibited a potential XO inhibitory activity with the molybdenum-pterin center domain as the active binding site. Salvianolic acid C, the active XO inhibitor, also exerted a potent hypouricemic effect on the mouse model induced by potassium oxonate. Such findings may be used to indicate the usefulness of the integrated approach for this scenario.

Tang, H. J., Yang, L., Li, W., Li, J. H., and Chen, J.,Exploring the interaction between Salvia miltiorrhiza and xanthine oxidase: insights from computational analysis and experimental studies combined with enzyme channel blocking, Rsc Advances, 2016, 6, 113527-113537.

The co-induced effects of molybdenum and cadmium on antioxidants and heat shock proteins in duck kidneys

Molybdenum (Mo) is an essential element for human beings and animals; however, high dietary intake of Mo can lead to adverse reactions. Cadmium (Cd) is harmful to health. To investigate the toxicity of Mo combined with Cd in duck kidneys, 240 ducks were randomly divided into six groups and treated with a commercial diet containing Mo, Cd or Mo combined with Cd. Kidneys were collected on days 30, 60, 90 and 120 for determining the expression of heat shock proteins (HSPs), including HSP60, HSP70 and HSP90 in the kidney through quantitative RT-PCR. We also determined the antioxidant activity indexes in the kidney mitochondria. Moreover, kidney tissues at 120 days were subjected to histopathological analysis with the optical microscope. The results indicated that the expression of HSPs was highly significantly (P < 0.01) upregulated in the kidneys of the combination groups and the Cd group. Exposure to Cd and a high dose of Mo decreased the total antioxidative capacity and the activity of xanthine oxidase, while malondialdehyde levels and the activity of nitric oxide synthase increased compared with those of the control groups in the kidney mitochondria. This was particularly evident at 90 and 120 days. Histopathological lesions included congestion and bleeding in the renal interstitium, swelling of the distal convoluted tubule epithelial cells, granular degeneration and blister degeneration in the renal tubular epithelial cells. These results suggest that a combination of Mo and Cd leads to greater tissue damage and has a synergistic effect on kidney damage. Oxidative damage of kidney mitochondria may be a potential nephrotoxicity mechanism of molybdenum and cadmium, and the high expression of HSPs may play a role in the resistance of kidney toxicity induced by Mo and Cd.

Xia, B., Cao, H. B., Luo, J. R., Liu, P., Guo, X. Q., Hu, G. L., and Zhang, C. Y.,The Co-induced Effects of Molybdenum and Cadmium on Antioxidants and Heat Shock Proteins in Duck Kidneys, Biological Trace Element Research, 2015, 168, 261-268.

Xanthine oxidase (EC and xanthine dehydrogenase (EC belong to the molybdenum hydroxylase flavoprotein family; they represent different forms of the same gene product. Xanthine oxidoreductase activity is rate-limiting in purine catabolism. The enzymes can metabolize xenobiotics, including a number of anticancer compounds, to their active metabolites.

Xanthine oxidoreductase is implicated in the pathophysiology of inflammatory diseases and atherosclerosis and in ischemia-reperfusion injury.

Pritsos, C.A., Cellular distribution, metabolism and regulation of the xanthine oxidoreductase enzyme system, Chem.Biol.Interact., 12-1-2000, 129, 195-208.

The role of molybdenum in xanthine oxidase has been extensively studied [Collison et al., 1996; Peive, 1968]. During the enzyme-catalysed reaction the oxidation state of molybdenum changes and so molybdenum is involved in the electron-transfer pathway. The substrate interacts directly with molybdenum. The molybdenum cofactor is an oxomolybdenum pterin complex similar to the cofactor of nitrate reductase [Berks et al., 1995; Campbell, 1996; Collison et al., 1996; Peive, 1968].

Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev., 1996, 25, 25.
Peive, Ya. V.,(ed.), Biol. Rol Molibdena, Sb. Tr. Simp. 1968 (Publ. 1972), Nauka, Moscow, U.S.S.R., 207, 235.
Berks, B. C., Ferguson, S. J., Moir, J. W. B. and Richardson, D. J., Biochim. Biophys. Acta - Bioenergetics, 1995, 1232, 97.
Campbell, W. H., Plant Physiology, 1996, 111, 355.
Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev., 1996, 25, 25.

Xanthine oxidizing enzymes isolated from leaves of leguminous plants did not react with molecular oxygen at a significant rate, indicating that all of them are xanthine dehydrogenase . The visible absorption spectrum of the pure protein was assigned: 312 - 390 nm, related to the molybdopterin component of the enzyme; 420 - 510 nm, flavin chromophores; 550 nm, iron-sulphur centres. The fluorescence excitation spectrum showed peaks at 274 and 368 nm, similar to pterin and xanthopterin. The fluorescence emission spectrum was characterized by two maxima at about 400 and 460 nm, typical of flavin chromophores.

Montalbini, P., Xanthine dehydrogenase from leaves of leguminous plants: Purification, characterization and properties of the enzyme, Journal of Plant Physiology, 2000, 156, 3-16.

Xanthine dehydrogenase (xanthine dehydrogenase) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The xanthine dehydrogenase gene has been isolated.

Pitts, R.J. and Zwiebel, L. J., Isolation and characterization of the xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata, Genetics, 2001, 158, 1645-1655.

The compound 1-(3, 4- dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine (PR5) is a member of a novel class of compounds, xanthine oxidase electron acceptor-inhibitor drugs. They have potential use in the prevention of free radical mediated tissue damage in organ ischemia-reperfusion diseases.

PR5 acts as an alternative electron acceptor in xanthine oxidase catalysed oxidation of xanthine. The action of PR5 is associated with the inhibition of superoxide radical formation. It is suggested that PR5 binds and becomes reduced at the molybdenum centre of the xanthine oxidase.

Dambrova, M., Baumane, L., Kiuru, A., Kalvinsh, I., and Wikberg, J. E., N-Hydroxyguanidine compound 1-(3,4-dimethoxy- 2-chlorobenzylideneamino)- 3-hydroxyguanidine inhibits the xanthine oxidase mediated generation of superoxide radical, Arch.Biochem.Biophys., 5-1-2000, 37 , 101-108.

The drug allopurinol is used to treat xanthine dehydrogenase-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of xanthine dehydrogenase. The 3.0 Angstrom resolution structure of the xanthine dehydrogenase -alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new xanthine dehydrogenase inhibitors.

Truglio, J.J., Theis, K., Leimkuhler, S., Rappa, R., Rajagopalan, K. V., and Kisker, C., Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus, Structure, 2002, 10, 115-125.

Reactive oxygen species, either superoxide anion radical or hydrogen peroxide, are generated when xanthine oxidase catalyses the hydroxylation of purines. Xanthine oxidase serum levels are increased in various pathological states: hepatitis, inflammation, ischemia-reperfusion, carcinogenesis and aging. The reactive oxygen species are involved in oxidative damage. The inhibition of this enzymatic pathway might be beneficial. Excess of uric acid, the metabolic product of xanthine oxidase, can lead to gout. Allopurinol is a clinically useful inhibitor used in the treatment of gout.The structure and mechanism of the enzyme, associated pathological states, and the development of new xanthine oxidase inhibitors are reviewed.

Xanthine oxidase inhibition

Xanthine oxidoreductase (XOR), which is widely distributed from humans to bacteria, has a key role in purine catabolism, catalyzing two steps of sequential hydroxylation from hypoxanthine to xanthine and from xanthine to urate at its molybdenum cofactor (Moco). Human XOR is considered to be a target of drugs not only for therapy of hyperuricemia and gout, but also potentially for a wide variety of other diseases. In this review, we focus on studies of XOR inhibitors and their implications for understanding the chemical nature and reaction mechanism of the Moco active site of XOR. We also discuss further experimental or clinical studies that would be helpful to clarify remaining issues.

Nishino, T. and Okamoto, K., Mechanistic insights into xanthine oxidoreductase from development studies of candidate drugs to treat hyperuricemia and gout, Journal of Biological Inorganic Chemistry, 2015, 20, 195-207.

Coumarin derivatives competitively inhibited xanthine oxidase. Esculetin (6,7-dihydroxycoumarin) had the highest affinity toward the binding site of xanthine oxidase due to the interaction of 6-hydroxyl with the E802 residue of xanthine oxidase. Esculetin was the most potent suppressor of reactive oxygen species. Esculetin was the most potent agent at protecting living cells against A beta-damage mediated by reactive oxygen species.

Lin, H.C., Tsai, S. H., Chen, C. S., Chang, Y. C., Lee, C. M., Lai, Z. Y., and Lin, C. M., Structure-activity relationship of coumarin derivatives on xanthine oxidase-inhibiting and free radical-scavenging activities, Biochemical Pharmacology, 2008, 75, 1416-1425.
Borges, F., Fernandes, E., and Roleira, F., Progress towards the discovery of xanthine oxidase inhibitors, Current Medicinal Chemistry, 2002, 9, 195-217.

An overview of the current state of our understanding of the reaction mechanism of the molybdenum-containing enzyme xanthine oxidoreductase is presented, with an emphasis on work done in the past five years. Recent studies of the biosynthesis of the pterin cofactor bound to the metal in the active site are also reviewed, as is crystallographic work that has clarified the coordination geometry of the molybdenum center. This structural work provides the context in which to understand recent mechanistic studies of the enzyme, in particular those aimed at elucidating the role of specific amino acid residues in the active site of the enzyme.

Hille, R., Structure and function of xanthine oxidoreductase, European Journal of Inorganic Chemistry, 2006, 1913-1926.

Acute lung injury represents a wide spectrum of pathologic processes, the most severe being the acute respiratory distress syndrome. Reactive oxygen intermediates have been implicated in the pathobiochemistry of acute lung injury. The endogenous sources that contribute to the generation of reactive oxygen intermediates in acute lung injury probably include, inter alia, the molybdenum hydroxylases. Gene expression of xanthine dehydrogenase/XO is regulated in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events such as hypoxia. Posttranslational processing is also important in regulating xanthine dehydrogenase/XO activity.

Hoidal, JR, Xu, P, Huecksteadt, T, Sanders, KA, Pfeffer, K, Sturrock, AB, Lung injury and oxidoreductases, Environmental Health Perspectives, 1998, 106, 1235-1239

Xanthine oxidase: mutations associated with functional disorder of xanthine oxidoreductase and hereditary xanthinuria in humans

Xanthine oxidoreductase (XOR) catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD(+) or O-2. The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR) due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors.

Ichida, K., Amaya, Y., Okamoto, K., and Nishino, T., Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans, International Journal of Molecular Sciences, 2012, 13, 15475-15495.

Xanthine oxidase mechanism of inhibition by flavonoids and gallic acid derivatives

The main active center of the molybdoenzyme xanthine oxidase is a molybdopterin buried in a cavity. One possible mechanism of inhibition is the attraction of an inhibitor molecule inside the cavity. It is important to understand the mechanisms of the enzyme inhibition to help in the search of new inhibitors. In this work the attraction was studied computationally by ab initio (DFT) calculations. Two properties were shown to be important in the design of new inhibitors of medium size derived from flavonoids: the molecule must be polar, with a longitudinal dipole moment, and must be weakly dissociated at physiological pH.

Xanthine oxidoreductase (XOR) is a molybdoflavoenzyme, which is abundant in cow’s milk. It appears in two interconvertible forms xanthine dehydrogenase (XDH), and xanthine oxidase (XO).

Xanthine oxidoreductase catalyzes the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. Oxidative hydroxylation occurs at the molybdenum center. With XDH NAD+ is reduced; with XO molecular oxygen is reduced at the flavin center.

Reduction of molecular oxygen produces free radicals which can damage tissues. XO is a potentiallydestructive agent in the vasculature.

Xanthine oxidase is a well-established target of drugs against gout and hyperuricemia.

Some pathological conditions show enhanced plasma XO levels: hepatic, acute viral infection, hemorrhagic shock, thermal stress,and hypercholesterolemia.

Allopurinol is applied as an inhibitor of xanthine oxidase but may induce hypersensivity reactions in patients with renal insufficiency and concomitant administration of thiazide diuretics. These undesirable effects have prompted efforts to isolate other types of XO inhibitors. Ethnobotanical research has provided XO inhibitors of natural origin.

Two main classes of molecules have been selected.

Flavonoids are natural compounds in plants, fruits, and vegetables. They are anti-oxidants. They are able to inhibit xanthine oxidase.

Gallic acid derivatives extracted from a Neo-Caledonian plant, Cunonia macrophylla, have been tested in vitro toward xanthine oxidase: gallic acid, ellagic acid, and ellagic acid-4-O-β-D-xylopyranoside.The last is the most active toward xanthine oxidase.

To evaluate the attraction inside the cavity, the electrostatic potential between the charged molybdopterin molecule and two series of inhibitors, flavonoids and some gallic acid derivatives, have been calculated using the multipolar development supplied by the Gaussian package. The good concordance between the electrostatic force and IC50 (the half maximal inhibitory concentration) shows that the attraction is an important factor in the inhibition and must be taken into account in the designing of new drugs.

Lespade, L. and Bercion, S., Theoretical Study of the Mechanism of Inhibition of Xanthine Oxidase by Flavonoids and Gallic Acid Derivatives, Journal of Physical Chemistry B, 2010, 114, 921-928.

Flavonoids interaction with molybdenum hydroxylases review

Molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, are metalloflavoproteins that catalyze both oxidation and reduction of a broad range of drugs and other xenobiotics indicating the importance of these enzymes in drug oxidation, detoxification and activation. Both enzymes are also involved in some physiological processes and also the metabolism of some endogenous compounds which may indicate their important roles in in vivo conditions. Superoxide radical and hydrogen peroxide produced during molybdenum hydroxylases-catalyzed reactions may be relevant in various disease conditions. Therefore, the interference with the function of molybdenum hydroxylases could be of great importance. Flavonoids are a large group of polyphenolic compounds that are able to interfere with xanthine oxidase and aldehyde oxidase function. As flavonoids are consumed in high content in our daily life, their potential to interfere with molybdenum hydroxylases could be a serious concern for consumer safety. However, the subject has not received enough attention and has usually been overshadowed by that of cytochrome P450 as the most important drug metabolizing enzyme system. The present review focuses on the different aspects of flavonoids interaction with molybdenum hydroxylases considering literature published mainly in the last 2 decades. The review also provides insight into some research areas that may offer a great potential for future studies.

Rashidi, M. R. and Nazemiyeh, H., Inhibitory effects of flavonoids on molybdenum hydroxylases activity, Expert Opinion on Drug Metabolism & Toxicology, 2010, 6, 133-152.

Substrate binding to molybdenum in the enzyme xanthine oxidoreductase rate limiting step

X-ray crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 angstrom and of the reduced bovine milk xanthine oxidoreductase2.1 angstrom, representing a reaction intermediate, are reported. The urate molecule is near the Mo ion, and high electron density connects them: hencea covalent link between molybdenum and urate via a bridging oxygen from urate. Thebridging electron density is bent, spanning a total of 3.5 Å, and connects to the C8 atom of urate, the position where xanthine becomes hydroxylated during the reaction.

The structure presents a picture of arrested catalysis at the step of the intermediate formed in the course of oxygen atom transfer from the molybdenum coordination sphere to the substrate carbon to be hydroxylated.

The rate-limiting step in the enzymatic catalysis is breaking of the Mo-O bond accompanied by electron transfer and followed by release of the product.

Okamoto, K., Kawaguchi, Y., Eger, B. T., Pai, E. F., and Nishino, T., Crystal Structures of Urate Bound Form of Xanthine Oxidoreductase: Substrate Orientation and Structure of the Key Reaction Intermediate, Journal of the American Chemical Society, 2010, 132, 17080-17083.

Copper inhibition of xanthine oxidase

Milk xanthine oxidase forms optically observable complexes with Cu2+ ion. Cu2+ ion binds to milk xanthine oxidase with sulfur and nitrogenous ligands. Two Cu2+ bound milk xanthine oxidase complexes are formed at two different time scales of the interaction, earlier than 5 ms and at around 20 s. The second complex may be responsible for the inhibition of the enzyme activity.

Sau, A.K., Mondal, M. S., and Mitra, S., Interaction of Cu2+ ion with milk xanthine oxidase, Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, 2001, 1544, 89-95.

Xanthine oxidase (XO)-catalyses nitrite reduction to nitric oxide under anaerobic conditions. The XO reducing substrates xanthine, NADH, and 2,3 -dihydroxybenzaldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism, Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions

Li, H.T., Samouilov, A., Liu, X. P., and Zweier, J. L., Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction - Evaluation of its role in nitric oxide generation in anoxic tissues, Journal of Biological Chemistry, 2001, 276, 24482-24489.

Nitrite reduction
Nitrite and nitrite reductases: from molecular mechanisms to significance in human health and disease

Nitrite, previously considered physiologically irrelevant and a simple end product of endogenous nitric oxide (NO) metabolism, is now envisaged as a reservoir of NO to be activated in response to oxygen (O2) depletion. In the first part of this review, we summarize and compare the mechanisms of nitrite-dependent production of NO in selected bacteria and in eukaryotes.

Bacterial nitrite reductases, which are copper or heme-containing enzymes, play an important role in the adaptation of pathogens to O2 limitation and enable microrganisms to survive in the human body.

In mammals, reduction of nitrite to NO under hypoxic conditions is carried out in tissues and blood by an array of metalloproteins, including heme-containing proteins and molybdenum enzymes.

In humans, tissues play a more important role in nitrite reduction, not only because most tissues produce more NO than blood, but also because deoxyhemoglobin efficiently scavenges NO in blood.

In the second part of the review, we outline the significance of nitrite in human health and disease and describe the recent advances and pitfalls of nitrite-based therapy, with special attention to its application in cardiovascular disorders, inflammation, and anti-bacterial defence.

It can be concluded that nitrite (as well as nitrate-rich diet for long-term applications) may hold promise as therapeutic agent in vascular dysfunction and ischemic injury, as well as an effective compound able to promote angiogenesis.

Castiglione, Nicoletta, Rinaldo, Serena, Giardina, Giorgio, Stelitano, Valentina, and Cutruzzola, Francesca, Nitrite and Nitrite Reductases: From Molecular Mechanisms to Significance in Human Health and Disease, Antioxidants & Redox Signaling, 2012, 17, 684-716.

Xanthine oxidase and purine metabolism

Xanthine oxidase is a molybdoflavoprotein enzyme with the composition 2FAD:8Fe:2Mo [Bray et al., 1996; Collison et al., 1996; Bray, 1963; Bray and Swann, 1972; Peive,1968]. The enzyme has a low specificity and will catalyse oxidation of many purines and aldehydes with very different rates of reaction. The oxidation of xanthine to uric acid is an essential stage in the catabolism of purine bases in some animals. For example, in parts of New Zealand where the concentration of molybdenum in the soil was low (0.03 ppm compared with 0.4 ppm in other areas) serious losses of sheep occurred owing to the formation of xanthine calculi in the kidneys of the sheep [Underwood, 1962]. Because of the low molybdenum concentration in the pasture xanthine oxidase activity was low and the conversion of xanthine to uric acid was hindered with the consequence that xanthine was precipitated in the kidneys. Raising the molybdenum level in the pasture prevented the development of renal calculi. The relationship between molybdenum concentration and xanthine oxidase activity is not, however, quite straightforward. At low concentrations molybdenum may stimulate the activity of xanthine oxidase; but at high concentrations molybdenum may actually reduce the activity of the enzyme. The activity of xanthine oxidase in cows' milk is proportional to the molybdenum content and both depend on the quantity of ingested molybdenum [Kiermeir and Capellani, 1957]. Addition of molybdenum to the diet causes an increase in the milk molybdenum but does not affect xanthine oxidase activity if this is high before feeding additional molybdenum.

The effect of molybdenum on xanthine oxidase activity in rats has been studied but the results are conflicting. Thus in one study increased dietary molybdenum (0-800 ppm), although causing an increase in liver molybdenum, caused a decrease in liver xanthine oxidase activity and in the concentration of uric acid in the blood [Cox et al., 1960]. With rats fed 1.0 mg Mo/kg body weight daily there were no changes in purine metabolism but 20 mg Mo daily caused increased xanthine oxidase activity in the liver and kidneys and in the blood urate and urea levels [Grigoryan et al., 1969]. According to another report rats receiving added dietary molybdenum (50-500 ug) showed increased xanthine oxidase activity and increased uric acid concentrations in the blood and urine, but 5 ug/day decreased xanthine oxidase activity [Peive, 1968; Gusev, 1969]. Xanthine oxidase activity rises markedly during virus multiplication and it is possible that the enzyme has a controlling effect on the pattern of nucleic acid synthesis in vivo [Bray, 1963].

Bray, R. C., in The Enzymes, eds Boyer, P. D., Hardy, L. and Myrback, K., Academic Press, New York, 2nd Edn., 1963, 7, 533.
Bray, R. C., Knowles, P. F. and Meriwether, L. S., in Magnetic Resonance in Biological Systems, ed. Ehrenberg, A., Malstrom, B. G. and Vanngard, T., Pergamon, 1967, 249.
Bray, R. C. and Swann, J. C., Structure and Bonding, 1972, 11, 107.
Bray, R. C., in Proceedings of the Climax International Conference on the Chemistry and Uses of Molybdenum, ed. Mitchell, P. C. H., Climax Molybdenum Co. Ltd, London and Ann Arbor, 1973, 216.
Bray, R. C., Bennett, B.m Burke, J. F., Chovnick, A., Doyle, W. A., Howes, B. D., Lowe, D. J., Richards, R. L., Turner, N. A., Ventom, A. and Whittle, J. R-S, Biochem. Soc. Trans., 1996, 24, 99.
Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev., 1996, 25, 25.
Peive, Ya. V.,(ed.), Biol. Rol Molibdena, Sb. Tr. Simp. 1968 (Publ. 1972), Nauka, Moscow, U.S.S.R., 207, 235.
Underwood, J. E., Trace Elements in Human and Animal Nutrition, Academic Press, London, 2nd Ed., 1962, 100.
Kiermeir, F. and Capellani, K., Naturwiss., 1957, 44, 69.
Peive, Ya. V., (ed.), Biol. Rol Molibdena, Sb. Tr. Simp. 1968 (Publ. 1972), Nauka, Moscow, U.S.S.R., 207, 235.
Cox, D. H., Davis, G. K., Shirley, R. L.and Jack, F. H., J. Nutr., 1960, 70, 63.
Grigoryan, M. S. and Brutyan, A. S., Tr. Erevan. Zootekh.-Vet. Inst., 1968, 29, 57, 61.
Grigoryan, M. S., Tatevosyan-Markaryan, L. G. and Asmangulyan, M. S. Grigoryan, L. G. Tatevosyan-Markaryan, and Asmangulyan, T. A., Biol. Zh. Arm., 1969, 22, 102.

The function in health and disease of the Mo-based enzymes sulfite oxidase, xanthine oxidase and aldehyde oxidase has been discussed. Xanthine oxidase, which occurs in the liver of humans and catalyses the formation of uric acid, may be anticarcinogenic through the development of protective systems against oxygen radicals.

Moriwaki, Y., Yamamoto, T., Higashino, K., Distribution And Pathophysiologic Role Of Molybdenum-Containing Enzymes, Histology And Histopathology, 1997,12, 513-524.

According to a molecular modelling study of the interaction of xanthine and hypoxanthine with xanthine oxidase correct positioning of the carbonyl group in the active site cavity is essential for a productive interaction with XO. The dimensions of the active site are mapped starting from the superimposition of the physiological substrates.

Rastelli, G., Costantino, L., Albasini, A., Model of the interaction of substrates and inhibitors with xanthine oxidase, Journal of the American Chemical Society, 1997, 119, 13, 3007-3016.
Doonan, C. J., Nielsen, D. J., Smith, P. D., White, J. M., George, G. N., and Young, C. G., Models for the molybdenum hydroxylases: Synthesis, characterization and reactivity of cis-oxosulfido-Mo(VI) complexes, Journal of the American Chemical Society, 2006, 128, 305-316.

A model system for molybdenum oxotransferases provides evidence for all biologically realistic intermediates, namely mononuclear Mo-VI, Mo-V and Mo-IV species. Dinucleation to EPR-silent [(Mo2O3)-O-V] species prevailing in homogeneous solution is suppressed by immobilising the active species to a polymeric support by two-point attachment.

Heinze, K. and Fischer, A., Polymer-supported dioxido-Mo-VI complexes as truly functional molybdenum oxotransferase model systems, European Journal of Inorganic Chemistry, 2007, 1020-1026.

Selenium-dependent molybdenum hydroxylases

Haft, D.H. and Self, W. T., Orphan SeID proteins and selenium-dependent molybdenum hydroxylases, Biology Direct, 2008, 3

Molybdenum hydroxylases drug-metabolizing ability

Drug metabolizing ability of molybdenum hydroxylases, which include aldehyde oxidase and xanthine oxidoreductase, and the variation of the activity amongst humans, with the highest activity, rats and mice and dogs are described in this review. Molybdenum hydroxylases, are involved in the metabolism of some medicines in humans. Interindividual variation of aldehyde oxidase activity is present in humans. Drug-drug interactions associated with aldehyde oxidase and xanthine oxidoreductase are of potential clinical significance.

Kitamura, S., Sugihara, K., and Ohta, S., Drug-metabolizing ability of molybdenum hydroxylases, Drug Metabolism and Pharmacokinetics, 2006, 21, 83-98.

Nitro-oleic acid – a novel and irreversible inhibitor of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 mu M, limiting both purine oxidation and formation of superoxide (O2-). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2-. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.

Kelley, E. E., Batthyany, C. I., Hundley, N. J., Woodcock, S. R., Bonacci, G., Del Rio, J. M., Schopfer, F. J., Lancaster, J. R., Freeman, B. A., and Tarpey, M. M., Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase, Journal of Biological Chemistry, 2008, 283, 36176-36184.

Density-functional theory models of xanthine oxidoreductase activity

The hydroxylation mechanism of the molybdoprotein xanthine oxidoreductase (XOR) has been modelled using density-functional theory. High activation barriers are often obtained for models of this enzyme due to the absence of factors that stabilize the accumulation of charge on the substrate at the transition state. Xanthine provides much lower barriers than small model substrates such as formamide or imidazole due to charge delocalization to centers which appear to interact with key residues in the protein. Of the two mechanisms of stabilization discussed in the literature-tautomerization and protonation of xanthine-density-functional theory calculations suggest that proton transfer from Glu1261 to N9 reduces the activation barrier by similar to 30 kcal mol-1 and leads to an intuitive product complex. Further, similar values for the activation barriers of methyl xanthine isomers lead to the conclusion that the wide variation in rates for substituted purines is due to interactions with key residues in the active site. In addition, the transition state for oxidation of xanthine can be superimposed over the X-ray structure of inhibitor-bound XO with high correlation suggesting that the enzyme active site orients the substrate into the ideal position for reaction. The activation barriers for models of a hypothetical tungsten-substituted XO are predicted to be similar to 10 kcal mol-1 higher in energy due to the higher reduction potential of the metal and unfavourable electrostatic interactions for the hydride transfer.

Bayse, C. A., Density-functional theory models of xanthine oxidoreductase activity: comparison of substrate tautomerization and protonation, Dalton Transactions, 2009, 2306-2314.
X-ray crystal structure of complex with xanthine and lumazine

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 angstrom resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 angstrom resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed

Pauff, J. M., Cao, H., and Hille, R., Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE, Journal of Biological Chemistry, 2009, 284, 8751-8758.

Cholesterol hydroxylation
Molybdoenzyme that catalyzes the anaerobic hydroxylation of a tertiary carbon atom in the side chain of cholesterol

Cholesterol is a ubiquitous hydrocarbon compound that can serve as a substrate for microbial growth. This steroid and related cyclic compounds are recalcitrant due to their low solubility in water, complex ring structure, the presence of quaternary carbon atoms, and the low number of functional groups. Aerobic metabolism therefore makes use of reactive molecular oxygen as co-substrate of oxygenases to hydroxylate and cleave the sterane ring system. Consequently, anaerobic metabolism must substitute oxygenase-catalyzed steps by O2-independent hydroxylases. Here we show that one of the initial reactions of anaerobic cholesterol metabolism in the beta-proteobacterium Sterolibacterium denitrificans is catalyzed by an unprecedented enzyme that hydroxylates the tertiary C25 atom of the side chain without molecular oxygen forming a tertiary alcohol. This steroid C25 dehydrogenase belongs to the dimethyl sulfoxide dehydrogenase molybdoenzyme family, the closest relative being ethylbenzene dehydrogenase. It is a heterotrimer, which is probably located at the periplasmic side of the membrane and contains one molybdenum cofactor, five [Fe-S] clusters, and one heme b. The draft genome of the organism contains several genes coding for related enzymes that probably replace oxygenases in steroid metabolism

Dermer, Juri and Fuchs, Georg, Molybdoenzyme that catalyzes the anaerobic hydroxylation of a tertiary carbon atom in the side chain of cholesterol, The Journal of biological chemistry, 2012, 287, 36905-36916.

Interaction of molybdenum with trypsin and pepsin

Molybdate binds to cationic centres of trypsin and pepsin enzymes.

Singh, R. P., Chaudhary, R., Rani, R., Kumar, S., and Arora, S. K., Interaction of Molybdenum With Trypsin and Pepsin by Dialysis Equilibrium Method, Asian Journal of Chemistry, 2010, 22, 1029.